Antimicrobial Heparin-binding Domains

Antimicrobial Digestion

Stomach enzymes produce antimicrobial peptides and intestinal enzymes inactivate heparin-binding domains of pathogens

The human digestive systems exhibits some amazing adaptations -- it can excise heparin-binding domains from ingested proteins and use them as antimicrobial peptides to inactivate ingested bacteria. Then in subsequent enzymatic steps in the intestines, the heparin-binding domains that otherwise could be used to adhere bacteria to the heparan sulfate proteoglycans of the intestinal lining, are chopped into inactive peptide fragments.

I was trying to find a cheap heparin-binding protein to use for a variety of research projects and finally found it in the form of whey lactoferrin. This protein is available in a relatively pure form for about $1 per milligram. Alternatively, I could buy it as a nutriceutical in 250 mg capsules for $0.50 per capsule or 1/500th the cost. My next step was to purify the cheap lactoferrin by binding it to chromatography beads with attached heparin. The lactoferrin stuck to the beads and other contaminating material was washed away. Finally, pure lactoferrin was released by increasing the salt concentration of the wash solution. My goal was to use proteolytic enzymes to hydrolyze the lactoferrin and produce peptide fragments containing heparin-binding domains. I naively pasted the known amino acid sequence of bovine lactoferrin into a website that would predict the cleavage locations of numerous proteases along the lactoferrin molecule. Amazingly, pepsin, the stomach protease, released a couple of peptides with heparin-binding domains, e.g. KCRRWQWRMKK, whereas trypsin, the intestinal protease, degraded all of the heparin-binding domains. I had a simple procedure for producing the peptides I wanted, but I also learned something about the beauty of the digestive system.

It took me a while to realize the utility of the alternative proteases. Production of heparin-binding peptides by pepsin enhances the sterilization of meals, because the heparin-binding domains are also generally antimicrobial. In fact, most antimicrobial peptides secreted by the skin or venom of a wide variety of organisms from primates to poison dart frogs have heparin-binding sequences. Degradation of heparin-binding domains by trypsin in the intestines is also advantageous, because numerous bacteria (e.g. E. coli O157;H7) and viruses (e.g. HIV and avian flu) use heparan sulfate regions of gut proteoglycans as receptors to immobilize these pathogens on the surface and initiate infections. Some toxins that rely on heparin-binding are also inactived in the intestines.

200th Post — Diet, Inflammation, Disease & Gut Flora

— all 200 Posts —

I started posting to Cooling Inflammation on 21 Aug, 2008 with How Your Diet Makes You Sick or Healthy.  My impetus for writing was my growing awareness that diet was the major reason why people were sick, and that health myths were preventing people from being healthy.  Inflammation originated by diet-inflicted injury and people attributed their sickness to genetics, environmental toxins and pervasive pathogens. 

My Path to the Obvious

My research background started with plant biochemistry, including carbohydrate structural analysis and polyphenol chemistry.  At that stage I was interested in understanding how plants protected (phytoalexins) themselves from pathogens, and I expected to use this perspective to explore human innate immunity.  From there, I went on to enzymology and protein characterization, biofilm structure, plant genetic engineering and breeding, monoclonal antibody production, mycotoxin detection, stem cell analysis, passive immunity in neonates, computational modeling of collagen and heparin binding, and heparan sulfate proteoglycan inhibition by inflammation.  These were temporary foci and the research imperatives, in retrospect, prevented me from seeing the bigger pictures, although they did leave me with a broad skill set.

Perspective: Water and Surface Tension

When I finally decided to slow down, smell the flowers and start having kids, I switched from research to teaching, from university to small liberal arts college.  For the first time, I actually thought about what I was teaching and my first revelation was that after teaching biochemistry for twenty years, I didn’t understand water and surface tension.  I could provide the platitudes from the Molecular Biology of the Cell, but I couldn’t do it mechanistically with colliding, sticky, energetic water molecules in my mind or at the blackboard.  I had to develop functional explanations of hydrogen bonds, entropy and thermal energy, that translated into the structuring of a layer of water molecules responsible for hydrophobic interactions and surface tension.  I extended that to include an explanation of the two layers of water holding together cytoplasmic membranes, the tube of structured water that holds together the cylinder of stacked bases in DNA or the shrink wrapping water layer surrounding proteins.

Perspective: Heparin Binding and Amphipathy of Sugars and Basic Amino Acids

As the kids got older, I started to dabble in research again and my expertise in carbohydrate chemistry led me into cartilage (mostly the glycosaminoglycan, GAG, chondroitin sulfate) synthesis and ultimately another GAG, heparan sulfate proteoglycans (HSPGs).  I was attracted to the dynamic HSPGs, that recycled with a half-life of six hours and formed layers around chondrocytes that secreted cartilage as they burrowed/ate through living cartilage.  I learned that the heparin filled granules of mast cells could be stained with berberine, which similarly stained the heparin in basement membranes of tissues and amyloids of Alzheimer’s, atherosclerosis and diabetes.  I was led by protein modeling of collagens to the binding of heparin to proteins and the revelation that basic amino acids (heparin binding domains) and sugars (heparin) are amphipathic, i.e. they have both hydrophobic and hydrophilic regions.  This is also true of plant polyphenolics.  Thus, polyphenolics, “basic” amino acids, “hydrophobic” amino acids, and sugars will all stack together.

Amphipathic Interactions

• DNA bases stack.
• Heparin binding sites of proteins are basic amino acids (Arg, Lys).
• Sugar binding sites in enzymes and lectins are hydrophobic amino acids (Trp, Tyr, Phe).
• Nuclear translocation signals, quartets of basic amino acids, bind to receptors with tryptophans.
• Tryptophans are the most highly conserved amino acids in the same proteins across great evolutionary distances.
• Hydrophobic bonding between tryptophan and a sugar or basic amino acid is ten times greater than hydrogen or ionic bonds.
• Tryptophan/Arginine ladders zip regions of proteins together.
• Polyphenols can disrupt cellular protein interactions by binding to receptors for carbohydrates/heparin, steroid hormones, amyloids, etc.
• Heparin holds dozens of hormones to receptors and changes the shapes of proteins, e.g. clotting and complement.
• Most nucleic acid binding proteins will also bind to the more negatively charged heparin.
• Bacteria use a pair of lysines to mark proteins for export.
• Peptides containing the basic amino acids of heparin binding domains (also produced by the specificity of gastric proteases) are antimicrobial, e.g. defensins, and so are plant polyphenols.
• Many drugs are active because they are domesticated plant polyphenols.

From Heparin Binding to Antigen Presentation

As soon as I realized that basic amino acids were involved in heparin binding, I started to look for the basic amino acids (R for arginine and K for lysine in amino acid sequences) in proteins known to bind heparin.  After study of hundreds of structures, it became obvious that heparin binding domains were simply a pair of basic amino acids (RR or KK or RK) with another within a distance of six amino acids.  No particular structure was necessary, as I later deduced, since binding to the heparin provided the structure.  In fact, in many X-ray crystallographic structures, the heparin binding regions on the surface of the protein are missing, because they are not in a defined shape.  I suspected that protein antigens involved in autoimmunity and allergy might be brought into cells for presentation to the immune system by interacting with HSPGs on the surface and so started to check them out for heparin binding domains.  I was very skillful at picking out pairs of Ks or Rs within sequences of hundreds of amino acids by that time, so I was shocked to see that the first dozen antigens that I checked, all had a triplet of basic amino acids.  I had discovered that autoantigens and allergens utilize a basic triplet analogous to the basic quartet used in nuclear translocation!  This also explained why proteins that interact with nucleic acids and are transported into the nucleus with a basic quartet are also prominent autoantigens.

Gut Flora and Immunity

Twenty years ago I read a curious description of leprosy that said that the course of infection could be either innocuous or devastating depending on whether the aggressive or the suppressive part of the immune system dominated.  I remained perplexed until I realized that diet and gut flora were the major determinants.  I was aware of the importance of diet at the outset of this blog, because it was clear that diet trumped genetics.  I was also aware thirty years ago in my studies of passive immunity, that milk contained bifidus factor, now known to be milk oligosaccharides, that controlled the growth of Lactobacilli that in turn controlled the development of the neonate immune system.  It was also known that bacteria-free mice had impaired immune systems.  It still took me several years for the relationship between diet, gut flora and immunity to make sense.  I began searching the literature for connections between gut flora and development of the immune system and soon noted experiments that linked filamentous bacteria with aggressive components and Clostridium spp. with Tregs.  A further refinement was linking resistant starch, a soluble fiber, with Clostridium.

My Current Views are Summarized in Three Health Diagrams

Diet, Gut Flora, Inflammation, Antigen Presentation, Tregs and Autoimmunity

Protein from the body and from food don’t normally stimulate the immune system, because there in no inflammation, the proteins lack basic triplets that enhance presentation, and antibody production and aggressive T cells are suppressed by Tregs.  Diet can throw the balance toward autoimmunity and allergy, by producing inflammation, e.g. hyperglycemia/AGE or high omega-6 fatty acids/prostaglandins, and starving gut flora needed for Treg production by eating processed food lacking soluble fiber.  The combination of inflammation and Treg deficiency causes proteins, either self or potential allergens, which have basic triplets to be presented to the immune system and stimulates attack by the immune system.

The Cure is to Cool Inflammation and Stimulate Tregs with Diet and Bacteria

I have provided an outline with The Anti-Inflammatory Diet to avoid inflammation, to stimulate existing gut flora with soluble fiber and encourage Treg production.  Mark Sisson, on Mark’s Daily Apple has provided an excellent dietary guide that also provides starch guidelines.  If you already have symptoms of autoimmune disease or allergies, then Richard Nikoley provides gut flora repair advice on Free the Animal, and Dr. B G provides more details on Animal Pharm.

Autoimmunity and allergies are not genetic destiny and they can be cured with diet and bacteria.

Insulin-like Growth Factor, Diabetes Autoantigen

IGF Binding to Heparin is Basis for Receptor Interaction, Internalization and Immunization

Examination of the protein sequence of insulin-like growth factors reveals strong heparin-binding domains (triplet of basic amino acids) that are also associated with internalization. Similar heparin internalization domains are also found on allergens and autoantigens. It was a small leap to expect that IGFs would also become autoantigens under inflammatory conditions that minimize heparan sulfate proteoglycan production.

Triplets of Basic Amino Acids Internalize Proteins

In several articles on this blog, I have discussed proteins that are internalized by their heparin binding domains. Heparin binding domains consistent only of a pair of basic amino acids, e.g. RK, flanked by one or more basic amino acids within a hydrophobic sequence of protein, are not sufficient to mediate internalization on heparan sulfate proteoglycans. A triplet of basic amino acids is usually required. Simple inspection of amino acid sequences is sufficient to identify these regions.

Internalization Triplet Identified in Insulin-like Growth Factor Binding Proteins

I noticed in a paper that insulin-like growth factors bind to epidermal growth factor receptors. I have previously written an article showing that EGF1 binds to its receptor via heparin, i.e. both the EGF and the receptor have heparin-binding domains. So I suspected that IGFs also had heparin binding domains. Inspection of the sequences readily identified simple heparin binding domains with pairs, but not triplets of basic amino acids. A search of the literature confirmed that heparin mediated IGF binding to receptors. A further search indicated that the heparin binding domains from proteins that bind and control the activity of IGFs could mediate internalization of proteins into cells and also into nuclei.

Internalization Triplets Are Associated with Allergens and Autoantigens

I have previously noted that all allergens and autoantigens have internalization triplets of basic amino acids. The presence of these triplets in IGF binding proteins suggested that IGF binding proteins might also be autoantigens. A quick check of the literature showed that antibodies against IGFs themselves frequently occur in type I diabetes. This suggests that the IGF-binding protein complexes are internalized and IGFs are immunologically presented during inflammation to produce anti-IGF antibodies. It is interesting that the other autoantigens for type I diabetes, e.g. transglutaminase, also have the expected internalization triplets.

references:
Maruyama T, Murayama H, Nagata A, Shimada A, Kasuga A, Saruta T.
Anti-insulin-like growth factor-1 autoantibodies in type 1 diabetes. Ann N Y Acad Sci. 2002 Apr;958:267-70.

Miao D, Yu L, Eisenbarth GS. Role of autoantibodies in type 1 diabetes. Front Biosci. 2007 Jan 1;12:1889-98.

Goda N, Tenno T, Inomata K, Shirakawa M, Tanaka T, Hiroaki H. Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5. Exp Cell Res. 2008 Aug 1;314(13):2352-61. Epub 2008 May 29.

Amyloids and Heparin

Amyloid proteins form fibers as they systematically stack in the presence of heparin. Examples of amyloids are the beta-amyloid and tau of Alzheimer’s disease, prions, and perhaps even sickle cell anemia fibers.

Amyloids are proteins that stack into fibers and can be detected microscopically by binding particular stains, e.g. Congo Red. All of the amyloid proteins are also heparin binding, e.g. Alzheimer’s amyloid is 50% heparin sulfate. I surmise that the Congo Red is binding to the heparin-binding domains, because Congo Red also binds to cellulose and a particular lipopolysaccharide from Shigella flexneri. Antibodies and enzymes that bind to polysaccharides, bind through tryptophan residues that are flat, hydrophobic plates with regular spacing. The amyloid fibers presumably bind the broadly spaced hydrophobic surfaces of Congo Red by providing corresponding complementary hydrophobic regions of the basic amino acids that are grouped on the stacked amyloids.

The interactions between carbohydrates, even highly charged polysaccharides such as heparin, and hydrophobic conjugated ring systems of aromatic molecules, such as dyes (Congo Red, Berberine) or aromatic amino acids (phenylalanine, tyrosine or tryptophan) may seem counterintuitive. The ring structures of sugars are actually hydrophobic on the relatively flat surfaces, surrounded by the hydrophobic edges of the hydroxyl groups. Tryptophans are amino acid-two ring systems that are the typical binding surfaces for sugars. The large, negatively charged sulfate groups on heparin sugar units limit the binding of aromatics, such as the dyes berberin or alcian blue to just one side of the units. Proteins bind to heparin by a combination of the positive charged at the ends and the hydrophobic chain of the basic amino acids, arginine and lysine. Thus, binding of a protein to heparin is a two step process of initial charge interactions followed by stronger hydrophobic interactions. Binding of the basic amino acids to aromatic amino acids can be observed in arginine/tryptophan ladders evident in F-spondin.

The cooperative of action of heparin in the formation of beta-amyloid, and many other experimental protein fibers has been thoroughly studied. Tau protein also forms intraneuronal fibers in Alzheimer’s, but a facilitating polysaccharide has not yet been identified. I have examined the protein sequence of the human tau protein and identified numerous heparin-binding domains. Also the X-ray crystallographic structure of tau has not be determined, because tau is considered an intrinsically unstructured protein. Many proteins with multiple heparin-binding domains are unstructured, because these proteins only reach a stable structure when they are bound to heparin. It is not likely that heparin is intracellular in healthy neurons, so either the integrity of neurons with tau fibers has been corrupted and heparin has entered, or the fiber enhancing molecule is a nucleic acid, e.g. RNA. There aren’t many other choices.

Knowledge of the rapid cycling properties of heparin may explain the toxic properties of amyloids. Amyloids are toxic when they are in contact with the cytoplasmic membrane surface. The contact toxicity is consistent with the amyloid fibers interacting with the heparan sulfate proteoglycans (HSPGs) embedded in the cytoplasmic membrane. It is known that HSPGs rapidly cycle between secretion and uptake. Both heparin and proteins with strong heparin-binding domains, e.g. protamine, HIV tat protein, are rapidly internalized. It is possible that amyloids bind to the uptake system, but the large size of the fibers blocks their internalization and paralyzes the uptake system. Paralysis of the membrane functions may lead to death of the neuron. Prions also appear to form amyloids with heparin and may be toxic for the same reason.

An analogous amyloid process leads to the death of the insulin-secreting beta cells of the pancreas, leading to type I diabetes. Insulin is made by chopping the middle and ends off of a pre-protein. The middle section that is excised has a strong heparin binding, internalization domain, so if insulin processing is blocked, is this the basis for autoimmunity in the presence of inflammation? Antibodies against the beta-cells keep killing them and maintaining the requirement for supplemental insulin. I think that aggressive anti-inflammatory treatment and blocking of the offending antibodies (or induction of tolerance) could permit the beta-cells to recover.

Structures displayed were captured from NCBI MMDB using CN3D.

Inflammatory Proteins Bind Heparin

Particular amino acid sequences mark a protein for secretion, binding to heparin, uptake and internalization into the nucleus.

You can tell a lot about a protein from the sequence of its amino acids. Basic amino acids (arginine and lysine) arranged in groups, for example, usually mean (if it is an extracellular protein) that a protein binds to heparan sulfate proteoglycans.

It seemed strange to me that heparin-binding was so simple when I tried to determine the rules for heparin-binding by looking at the structures of several hundred proteins known to bind to heparin. Since heparin is heavily sulfated and the sulfates are negatively charged, at first I just color-coded the positively-charged , basic amino acids (blue) to look for oppositely charged heparin-binding sites on the surface of the proteins. Obvious blue patches were found on the surfaces of all of the proteins that bound to heparin and scattered blue spots were on the surfaces of other proteins. Moreover, similarly color-coded amino acid sequences showed that the blue patchs almost always had pairs of basic amino acids flanked within six amino acids by a third basic amino acid, i.e. BBxxxxB, where B is either arginine (R) or lysine (K) and x is a hydrophobic amino acid. It was surprisingly simple.

I was shocked at the simplicity, because most binding sites are made up of parts of regular secondary structures of helices or pleated sheets. If there were basic amino acids on these structures, which bound to heparin on one side, then the R/K would be repeated at specific intervals. For a helix, for example, the repeat would be BxxBxxB, because it takes three amino acids to return to the same side as the amino acids wind around in the helix. For the pleated sheet, the amino acids alternate on each side of the sheet, so the pattern is BxBxB. I found these kinds of heparin-binding domains also. The hardest patterns to find from sequences are groups formed as R/K’s on neighboring helices or sheets are brought together in the final folding of the protein.

One of the reasons that the simple pair plus one (BBxxxxB) was found so easily, is because the sequence is typically found on coils that only take shape in the presence of heparin. Thus the rigid binding of the domains to heparin is a result of the shape of the protein induced by the heparin. A related example of this phenomenon is the facilitation of the formation of amyloid fibers in the presence of heparin. The beta amyloid of Alzheimer’s disease for example, consists of a stack of small amyloid peptides with basic amino acids that line up and bind heparin along the length of the stack. Heparin is also an essential component in the amyloids of diabetes. Prions also seem to involve heparin. It is assumed that the cytoplasmic tau fibers of Alzheimer’s disease also have a similar facilitating polyanion (if not heparin), but it has not been identified.

Because of the essential nature of HSPG recycling, it is interesting that amyloid formation is toxic when the amyloid is in contact with cells. Perhaps the amyloid paralyzes HSPG recycling and thereby kills the cells. Treatments that disrupt amyloid binding to heparin, e.g. methylene blue, spare the neurons. This would also suggest the utility of berberine, a fluorescent dye for heparin, which is also a common herbal cure for arthritis, in treatment of many amyloid diseases.

The pair plus one is the minimal grouping of R/K’s that binds heparin, but larger groups bind more strongly and increase the complexity of the interaction between proteins and a cell. A triplet of R/K’s results in a protein binding to the heparan sulfate proteoglycans (HSPGs) on the surface of a cell, but as the HSPGs are recycled by being brought into vesicles within the ce)ll, the bound proteins are also internalized. These internalized proteins are then fused with lysosomes and the proteins are at least partially degraded by proteases. The proteins were released from the HSPGs by the degradation of the heparan. The modified proteins have a variety of fates. Some return to the Golgi for secretion, e.g.HSPGs and heparanase, whereas others are degraded in proteosomes and presented as potential antigen fragments on surface receptors, and still others are are transported to the nucleus. Those proteins transported into the nucleus have four R/K’s or to neighboring pairs of R/K’s, e.g. HIV-TAT, heparanase and transglutaminase 2 (?) Heparanase is intimately involved in cancer proliferation and transglutaminase is involved in Celiac and inflammation.

I have reproduced below the sequences of several human proteins from the National Center for Biotechnology Information. For simplicity, I have deleted the “uninteresting” amino acids between the heparin-binding domains. You will also see an occasional negatively charged amino acids (D/E) within the R/K groups and their hydrophobic neighbors. These amino acids bind to the amino sugars of the heparin.

transglutaminase 2
M---REKLVVRR---KFLKNAGRDCSRR---RRWK---KIRILGEPKQKRK

heparanase
M---REHYQKKFKNSTYSR---KLLRKSTFKNAK---RRKTAKMLKSFLK---RPGKK---KKLVGTK---KRRKLR

Tat [Human immunodeficiency virus 1]
M---KCYCKK---RKKRKHRRGTPQSSK---KEQKKTVASKAER

Chain A, Interleukin- 1 Beta
A---KKKMEKRFVFNK

lactoferrin
M---RRRR---RNMRKVR---RRAR---KGKK---KRKPVTEAR

Watson Makes Us Sick

Common Textbook: Molecular Biology of the Cell, Lacks Coverage of Critical Molecular Interactions

One of the major reasons why healthcare practitioners are unable to cure diseases, is that their molecular view of disease is outdated. Their models of key signaling interactions lack critical molecules and fundamental types of chemical bonds are ignored.

The Major Textbook Used to Train Medical Students Lacks Essential Cellular Interactions

The most pervasive and perhaps the best text book on cell biology, The Molecular Biology of the Cell, first authored by James Watson, lacks a discussion of the bonding of aromatic amino acids (tryptophan, tyrosine, phenylalanine) with basic amino acids (arginine, lysine), carbohydrates, and aromatic phytochemicals, e.g. plant antioxidant or alkaloids. As a result, medical school graduates lack familiarity with the prominent interactions that dominate disease and drug treatments.

Hydrophobic Bonding to Aromatic Amino Acids Dominates Cell Molecular Biology

The dominating significance of aromatic hydrophobic bonds is the strength of these bonds, ca. 20 kcal/mol versus, the commonly considered weak bonds (hydrogen, ionic) at 1-2 kcal/mol, the same as the kinetic energy of water at body temperature. Thus, structures, such as alpha helices and beta sheets of proteins, require multiple weak bonds to be stable, but the hydrophobic bonding of tryptophan to a single arginine draped across its surface is stable.

Examples:

Tryptophan is the most highly conserved amino acid in protein structures (more than cysteine forming disulfide bonds!). This means that tryptophan is the most important amino acid in protein structure, and probably determines how proteins fold.

Carbohydrates have hydrophobic faces to their ring structures and typically bind to lectins, glycosidases and glycanases, via the hydrophobic surfaces of tryptophans or tyrosines in active sites.

Transport of proteins into nuclei is by binding of arginine or lysine residues of nuclear localization signals (basic quartets or neighboring basic pairs) to tryptophan hydrphobic residues projecting from the surface of LRR (leucine-rich repeat) importin molecules.

Heparin binds to basic amino acids in proteins via hydrophobic interactions. Aromatic dyes, such as berberine, bind to heparin through similar hydrophobic interactions.

Heparin binds to the basic amino acids arrayed in stacks of amyloid molecules and berberine blocks these interactions. Congo Red, a diagnostic dye for amyloids, is an aromatic molecule. Similar interactions occur with prions and the plaques of atherosclerosis.

Acidic polysaccharides form the matrix of biofilms. Heparin and nucleic acids can also serve this function. PEG, which disrupts hydrophobic interactions, can be used to disrupt binding of proteins to heparin, nucleic acids and biofilm polysaccharides.

Heparin binding mediates the interaction between most growth factors or cytokines and their cell surface receptors.

Many viruses and bacteria bind to cell surfaces via heparan sulfate.

LDL binds to LDL receptors via heparan sulfate. ApoE in diagram (arg and lys in blue, hydrophobic in pink.)

Antimicrobial peptides, e.g. defensins, have groups of basic amino acids. Heparin binding domains excised from proteins as peptides are antimicrobial.

Stomach proteases cleave around heparin-binding domains to produce antimicrobial peptides. Intestinal proteases cleave within heparin-binding domains and inactivate bacterial and viral agglutinins.

Life starts with heparin, i.e. heparin is leaked into fertilized eggs to remove the small, highly basic proteins used to package the sperm chromosomes.

Heparin is injected experimentally into nerves to silence IP3 signaling based on the binding of the hydrophobic face of inositol to basic amino acids, similar to heparin binding domains, of the IP3 receptors located on the surface of the ER.

The cytoplasmic domains of some receptor proteins have basic regions that interact with the IPs of the membrane surface, but subsequently serve to transport membrane-derived vesicles to the nucleus via importin carriers.

Heparin/heparan sulfate proteoglycans are secreted bound to basic molecules such as polyamines or histamine.

Heparan sulfate proteoglycans are continually secreted and taken up with a half life of six hours. This circulation is a major transport system of most cells. Amyloid/heparan aggregates on the surface of nerves and gliadin/tTG/antibody/heparan complexes on endocytes (celiac) may poison this system.

All allergens and autoantigens have a triplet of basic amino acids that may be involved in the initial aberrant presentation of these antigens as a result of the internalization by the carbohydrate-binding domain of mannose receptors on the surface of inflammation-stimulated immune cells.

Many neurotransmitters bind to their receptors via hydrophobic, aromatic interactions. These same receptors interact with hydrophobic, aromatic phytochemicals, e.g. “anti-oxidants.” Many spices, herbs, alkaloids and other phytochemicals have their abundantly complex interactions via these mechanisms.

Crystals of the tryptophan repressor involved in binding tryptophan and altering the expression of genes involved in tryptophan synthesis, shatter in the presence of tryptophan -- the tryptophan (yellow) strongly binds to basic amino acids (blue) in the tryptophan-binding domain of each repressor protein in the crystal and alters its shape.

Heparin and Inflammation

Heparin dominates the extracellular world and controls inflammation

Why is heparin such a big deal in inflammation? Heparin controls the communication between cells and to a great extent it also controls what goes in and out of cells. At the same time there appears to be a feedback system, so that cells that have their inflammation program triggered also change their production of heparin.

I am using the term “heparin” very loosely here to include all of the different forms of the highly sulfated polysaccharide. All of these different forms start as polysaccharides (long sugar chains) extending from proteins, i.e. proteo (protein) glycans (polysaccharides). The sugar chains are made by enzymes that alternately add a negatively charged sugar (glucuronic acid) and then a positively charged sugar (N-acetylglucosamine) in long chains to a four sugar linker attached to a protein. The “heparan” polysaccharide is then altered chemically and highly negatively charged sulfate groups are added. The alterations are not uniform along the length of the heparan, but rather form islands along the chains with special structures. In some cells, the long heparan sulfate chains are attacked by enzymes, so that the heavily sulfated islands are released as short chains, oligosaccharides, called heparin. Heparin is commonly secreted by mast cells as these cells secrete their companion molecule histamine during responses to allergens. Thus, one function of heparin, which is negatively charged, is to neutralize the positive charge on histamine as they are stored in vesicles prior to release.

Heparan sulfate proteoglycans (HSPGs) are continually secreted and then brought back into cells. This turnover is fairly rapid, so the HSPGs that dominate the surface of cells is renewed every six hours. This is true for most cells, including your cartilage cells, chondrocytes, that live in small HSPG-lined capsules within the cartilage (collagen fibers in another sulfated polysaccharide, chondroitin sulfate) as they mine the cartilage as their source of protein and carbohydrates at one end of the cell and synthesize new cartilage from the other end. Damaged cartilage must be pressed very firmly together, so that these chondrocytes can knit the damaged regions back together with their burrowing/synthesis action. At the same time that cartilage protein fibers and polysaccharides are being recycled on a tissue-wide scale (collagens have a lifetime of at least decades), the cells rapidly treadmill their HSPGs.

Some proteins travel on the HSPGs from one cell, to adjacent cells. The HIV protein called TAT is secreted from HIV-infected cells bound to HSPG. As the bound TAT is swept from one end of the cell to another, it encounters the HSPGs sweeping over the surface of neighboring cells and jumps ship, so to speak. The TAT is then swept into the unsuspecting neighbor that has not previously experienced HIV. The particular heparin-binding regions of the TAT protein are similar to the nucleic acid-binding regions of cellular proteins, so the TAT is transported to and into the nucleus, where the TAT acts as a transcription factor and prepares the cell for HIV infection.

Heparan sulfates dominate the extracellular region surrounding a cell and the heparan sulfate chains attach to both hormones and hormone receptors and mediate signaling. For example, the inflammatory and anti-inflammatory cytokines and their receptor proteins embedded in the cell surface all have heparin-binding domains. Heparin-binding domains are also present in proteins of the clotting system, most of the complement components and in many of the proteins that regulate development. Most defensive peptides with antimicrobial properties have heparin-binding domains and if heparin binding domains are removed from proteins, they are antimicrobial peptides.

Heparan sulfate proteoglycans dominate the extracellular interactions the way nucleic acids dominate the nucleus. Phospholipids and inositol phosphates may be similarly dominant in the cytoplasm. It is interesting that the active component in digestive fiber is inositol hexaphosphate, phytic acid. Also note that amyloid diseases, such as Alzheimer’s and type I diabetes are characterized by extracellular fiberous aggregates of proteins, e.g. beta-amyloid, on a scaffold of heparin. Some of the amyloid proteins don’t have heparin-binding domains, until they stack into fibers. The cellular scaffold for tau fibers has not yet been identified, but perhaps it is a form of polymerized inositol phosphate.

Many drugs act on heparin-binding or interacting domains and that is another reason why heparin and heparin-binding are critical elements of inflammation.

Toulouse-Lautrec

Cathepsin K and Heparin

The power of simple sequence pattern analysis in predicting protein behavior is illustrated in the case of Cathepsin K, a papain-like cysteine protease, involved in many degenerative diseases, bone development and Pycnodysostosis (Toulouse-Lautrec syndrome.) Triplets of basic amino acids are typical of heparin binding proteins that are internalized.

I admit that I am obsessed with inflammation and heparin. My daughters automatically yell out “Give him heparin!” when a patient on ER has a severe migraine attack. They think it is a good joke until the savvy doc reveals the latest approach and actually injects heparin with satisfying results. Heparin and inflammation are intimately involved and I predict that blood tests that determine the quality and quantity of protein-bound heparin, will ultimately be used as measures of chronic inflammation, as well as revealing a variety of diseases.

I have a habit of examining the molecular basis of diseases that I encounter on TV, in newspapers or in books. Wikipedia is my first source, followed by the National Center for Biotechnology Information (NCBI). As soon as I find the genes/proteins involved, I check to see if the structures has been determined by X-ray crystallography or NMR, and then I look at the amino acid sequence. I check for pairs or triplets of basic amino acids. Invariably the pairs are matched with a neighboring basic amino acid, and that is a putative heparin-binding domain. Triplets almost always indicate secreted proteins that are brought back into cells dependent on strong affinity for recycled heparan sulfate proteoglycans. Within minutes of hearing about a new disease, I usually know something about the molecules involved and particularly whether or not inflammation is going to be a major factor.

I was just reading Outlander by Diana Gabaldon and one of her characters has the short stature and disablity of Toulouse-Lautrec syndrome. I literally ran to my computer, because I am particularly interested in diseases of cartilage and bone. Since TLS is a genetic disease, I checked the NCBI Online Mendelian Inheritance in Man (OMIM) site and found that the genetic defect is in the cathepsin K gene. Cathepsin K is a protease similar to papain, which is intimately involved in many different facets of development, as well as cartilage and bone production. The cells that degrade cartilage to remodel bone, osteoclasts, use cathepsin K to degrade collagen.

I found a structure for cathepsin K bound to chondroitin sulfate. The structure looked all wrong, based on my prejudices -- the sugars of the polysaccharide should have been bound to the basic amino acids or to surface aromatic amino acids. The accompanying amino acid sequence told the whole story:

---DYRKKGYVTPVKNQGQCGSCWAFSSVGALEGQLKKKT---

There were two triplets of basic amino acids (R, arginine or K, lysine), indicative of internalization and strong heparin binding. I performed a quick literature search for heparin binding and internalization and found a reference that confirmed my hunches (note the title):

Nascimento FD, Rizzi CC, Nantes IL, Stefe I, Turk B, Carmona AK, Nader HB, Juliano L, Tersariol IL. Cathepsin K binds to cell surface heparan sulfate proteoglycans. Arch Biochem Biophys. 2005 Apr 15;436(2):323-32.

The article demonstrated that cathepsin K bound only to the surface of cells that produced heparin sulfate and was internalized only by heparin-producing cells. Moreover, cathepsin K changed shape as it bound to heparin, but not to chondroitin sulfate.

This story underscores the predictive power of simple generalizations derived from the dominating interactions between heparin and proteins. Heparin-binding domains, because of their positive charges, stay on the surface of the protein, don’t tend to fold well into helices or other secondary structures and are readily recognized in amino acid sequences of proteins. Stronger heparin-binding domains involved in internalization or transport into nuclei are even more stereotyped as triplets or quadruplets, respectively, of basic amino acids.

There are some complicating special cases involving basic amino acids, since these amino acids are also involved in glycosylation, nucleic acid binding, inositol phosphate interactions, phospholipid interactions, protein folding/chaperone binding, and protease action, but the generalizations outlined here provide a starting point for exploring the exciting area of heparin binding.

Knowing just a few typical patterns, you can just look at a protein sequence and amaze people by telling them that they should be able to purify their protein on heparin Sepharose! You can also point and gasp at the fact that in the early 1990’s in China the bird flu hemagglutinin picked up a new sequence with a quartet of four basic amino acids. When I saw that I called the CDC and explained the new cell receptor! It still has me scared -- am I the first one to notice the potential for a pandemic much more severe than the 1918 Spanish flu?

Lyme Spirochete Binds to Heparan in Blood Vessels

Borrelia burgdorferi Sticks to Host Cells via Heparin-binding Proteins

A research group at the University of Calgary has watched the binding of fluorescent Borrelia burgdorferi spirochetes, the Lyme disease pathogen, to the surface of blood vessels in mice. (ref. below) A small heparin molecule was shown to block this interaction between the spirochete surface protein BBK32 and the heparan sulfate proteoglycans of the endothelial cells of the skin blood vessels.

The heparin-binding domains in the spirochete protein, BBK32 are easy to spot in the amino acid sequence of this protein that I downloaded from the NCBI protein database:

>gi|19072701|gb|AAL84596.1| BBK32 [Borrelia burgdorferi]
MKKVKSKYLALGLLFGFISCDLFIRYEMKEESPGLFDKGNSILET
SEESIKKPMNKKGKKIARKKGKSKVSRKEPYIHSLKRDSANKSN
FLQKNVILEEESLKTELLKEQSETRKEKIQKQQDEYKGMTQGSL
NSLSGESGELKETIESNEIDITIDSDLRPKSSLQDIAGSNSISYTDE
IEEEDYARYYLDEDDEDDEYYEDDYEEIRLSNRYQSYLEGVKYNV
DSAINTINKIYDTYTLFSTKLTQMYSTRLDNLAKAKAKEEAAKFTK
EDLEKNFKTLLNYIQVSVKTAANFVYINDTHAKRKLENIEAEIKTL
IAKIKEQSNLYEAYKAIVTSILLMRDSLKEVQGIIDKNGVWY
Basic amino acids are K=lysine, R=arginine

The minimal heparin binding pairs, e.g. KKVKSK are shown in red and the strong heparin-binding triplets, e.g. KRK, are shown in blue. Notice that one triplet is augmented with several pairs to further enhance heparin binding.

I would also expect that BBK32 would be internalized into host cells and transported into the nucleus, where it may alter transcription, a la HIV-TAT. The existence of multiple, strong heparin-binding domains may also serve to bind the BBK32 (or the spirochetes) to multiple different heparan sulfate proteoglycans and interfere with the HSPG circulation system. This may have a toxic effect.

reference:
Norman MU, Moriarty TJ, Dresser AR, Millen B, Kubes P, Chaconas G. Molecular mechanisms involved in vascular interactions of the Lyme disease pathogen in a living host. PLoS Pathog. 2008 Oct 3;4(10):e1000169.

Bacterial Amyloid (Curli Fibers) Forms Biofilms

E. coli Curli Stacks in Congo Red Staining Fibers

We can’t cure diseases, because we don’t understand basic chemistry (what is hydrophobic) and biology (which came first the biofilm or the bacterial cell wall?)  Let’s look at a fundamental biological process, how bacteria form biofilms.

Biofilm Formation from Secreted Proteins and Polysaccharides

Investigators passed some E. coli through a special slide chamber so they could watch at high magnification as a single bacterium attached to the surface, divided to produce a colony of a few bacteria and then began to secrete proteins (curli fibers) and polysaccharides (colanic acid and cellulose) to make the biofilm matrix.  The matrix stained red with Congo Red.

Congo Red Stains Amyloids, Cellulose and Rare LPS



Staining with Congo Red shows that the spacing of hydrophobic patches on the surface of the biofilm matrix matches the flat hydrophobic, aromatic rings of the dye, Congo Red.  This particular dye is important, because Congo Red also specifically stains amyloid, e.g. beta amyloid of Alzheimer’s disease.  But Congo Red also binds to cellulose, a linear beta 1,4-glucan polysaccharide.  This seems paradoxical, because we are taught that the sugars of which a polysaccharide are made are hydrophilic.  That turns out to be a half-truth. 

Faces of Sugars Are Hydrophobic

The hydrogen bonding hydroxyl groups that make sugars water soluble and hydrophilic, radiate from a ring of carbons, and the faces of that ring cannot make hydrogen bonds.  The faces of sugars are hydrophobic and in most cases will bind to hydrophobic surfaces, such as aromatic amino acids, e.g. tryptophan, tyrosine and phenylalanine.  Thus, carbohydrate binding enzymes, such as shown in the figure bind cellulose (in grey and red) in a groove lined with aromatic amino acids (yellow and orange) so that each sugar orients over and sometimes sandwiched between aromatic amino acid residues.  This also explains why Congo Red binds to cellulose, since the aromatic rings of the dye bind to neighboring glucose residues along the relatively flat cellulose strand.  Most other polysaccharides and smaller sugars lack this spacing of sugars and they don’t stain red with Congo Red.

Basic Amino Acids Bind Hydrophobically

Another misperception is that basic amino acids, positively charged lysine and arginine, are hydrophilic.  The nitrogen atoms that make these amino acids positively charged, can form hydrogen bonds, but the hydrocarbon tails that have these nitrogenous tips, are hydrophobic.  Thus, basic amino acids and aromatic amino acids can stack to form tryptophan/arginine ladders in which they alternate.  A prominent example of these interdigitations are the way that nuclear localization signals, a quartet of basic amino acids, bind to importin via its projecting, spaced tryptophans and drag proteins through pores into the nucleus.  Similarly, the basic amino acids of heparin-binding domains extend across the hydrophobic faces of the sugars of heparin and hydrogen bond with their tips to the sulfates of the heparin.  In each of these binding examples the binding is primarily hydrophobic.

Amyloid Binds Congo Red by Stacked Heparin-Binding Domains

Amyloids are proteins that stack together like stacking chairs, so that each protein is oriented in the same way all along the stack.  In the case of the beta amyloid that makes up the toxic plaque in Alzheimer’s disease, each amyloid peptide is stacked like a hair pin on top of the next to make a fiber.  At the bend in beta amyloid, is a basic amino acid and the amyloid fiber has a band of basic amino acids along its length.  The spacing between the basic amino acids in an amyloid stack is just spanned by Congo Red, so amyloids are diagnostically stained red.  This same spacing of basic amino acids fits the sugars in heparin.  Thus, heparin can catalyze amyloid formation and is abundant in amyloid plaques in Alzheimer’s

Bacterial Biofilms Form from Amyloids and Polysaccharides

The E. coli cells that formed the biofilms that started this article secrete a protein, curli, that stacks as an amyloid into fibers.  These fibers stained by Congo Red and bind to the cellulose that is also produced by the E. coli.  It should not be surprising that biofilm formation is catalyzed by heparin and biofilm formation is a major problem in catheter infection, since heparin is used to coat catheters to keep them from forming blood clots.  Amyloids are also formed from stacked seminal acid phosphatase proteins that form fibers in the presence of heparin and facilitate HIV infection.

Do Biofilms Foment Amyloid Production?

Basic amino acids, sugars, aromatic amino acids and plant phytochemicals all bind each other via their hydrophobic surfaces.  It would not be surprising that bacteria that produce proteins and acidic polysaccharides that interact hydrophobically would also interact with host molecules with a similar spacing of hydrophobic surfaces, which are common in heparin-binding interactions and nucleic acid interactions.  The bacteria in biofilms produce both proteins and polysaccharides that may catalyze amyloid production.  The acidic biofilm polysaccharide, colanic acid, may replace heparan sulfate and curli should bind to heparin.

Berberine Binds Heparin and Blocks Amyloids and Biofilms

Just as bacterial products may compete for host heparin and heparin-binding domains, phytochemicals that interact with heparin, such as the phytochemical berberine, should disrupt heparin mediated molecular interactions, and by extension also biofilms.  There is experimental evidence for berberine both disrupts amyloid formation and biofilm assembly.

The Cause of Allegies and Autoimmune Diseases

Keyhole Limpet Hemocyanin (KLH): Internalized Antigen

Scanning the literature for a common protein that can be used as an experimental antigen, it becomes quickly obvious that a favorite is KLH. This would seem to be an odd choice -- why a keyhole limpet protein? But that is the wrong question.

Why is KLH such a good antigen, i.e. why is it readily presented to the host immune system? If you have been reading my posts, you might be thinking about triplets of basic amino acids and that is the answer.

As soon as I remembered the prominent use of KLH as an antigen, I checked the NCBI protein database and immediately found an unusual KKK (triple lysine) near the amino terminus of hemocyanin II ( it comes in two pieces). This triplet explains why KLH is such a good experimental antigen, because it is internalized into antigen presenting cells by its strong heparin-binding domain. Other components, adjuvants, are typically added to the KLH for injection to make sure that a strong local inflammation occurs.

Autoantigens Have Strong Heparin-Binding Triplet

I also learned that Hashimoto’s thyroiditis is an autoimmune disease mediated by the autoantigen thyroid peroxidase. A quick search reveals that thyroid peroxidase is an autoantigen, because it also has a triplet of basic amino acids that can enhance presentation under inflammatory conditions. Grave’s disease of hyperthyroidism is an autoimmune disease in which the thyroid receptor (with a basic triplet) is an autoantigen. The same kind of triplet of basic amino acids was found when I searched today for fire ant antigens and mosquito antigens.

I have also looked for the triplets in protein databases. The triplets are rare in cytoplasmic and extracellular proteins. The proteins that have triplets are usually identified as autoantigens in some disease. The triplets are common in nuclear proteins, since heparin-binding and nucleic acid-binding share the same basic amino acid domains. The nuclear internalization signal also results in rapid cellular internalization, e.g. HIV-TAT, heparanase, IGF-binding proteins. Nuclear proteins are common autoantigens in lupus.

Inflammation Plus Heparin-Binding Internalization: Allergy, Autoimmunity

Chronic inflammation can produce antibodies against proteins (foreign or self) with strong heparin-binding domains (triplets or sometimes neighboring pairs of basic amino acids, lysine or arginine). The generalization explains why particular proteins in pollens, foods, insects, pets, mites, asthma, MS, lupus, celiac, etc. produce antibody responses.

Bee Sting Allergy

Typical. I started to write an article on leukotrienes, the inflammatory derivatives of the omega-6 fatty acid, arachidonic acid, but ran across another powerful example to test my hypothesis to explain the cause of allergies. The leukotriene article will have to wait till another day.

Wikipedia is my source of choice for up-to-date summaries of biomedical information. I queried “leukotrienes” and immediately ran across the original name for these inflammatory compounds, “slow reacting substance of anaphylaxis”. I was initially distracted by the classic experimental use of snake venom and histamine to induce leukotriene production. Snake venom has the same enzyme, PLA2, as brown recluse spider venom (subject of a previous article) and honey bee venom, that releases arachidonic acid (ADA). ADA is an omega-6 fatty acid that is the starting material for inflammatory prostaglandins and leukotrienes.

The mention of honey bee venom in the Wikipedia article on anaphylaxis sent me on a quick check of the structure and sequence of the honey bee allergen. I initially found that the major allergen is a hyaluronidase. I quickly searched for a three amino acid sequence that I predicted would make it an allergen. It was just where I expected to find it. About two thirds of the way along the amino acid sequence I found, -TTSRKKVLP-. Three basic amino acids together, in this case -RKK-, argininine-lysine-lysine, form a strong heparin-binding domain, that I believe takes proteins into cells and during inflammation primes the immune system for allergic responses.

I have found the same strong heparin-binding domain associated with allergens of ragweed, dust mites and peanuts. The principal autoantigens of autoimmune diseases, such as lupus, celiac, etc. also display the same unusual sequences. In lupus, or example, nuclear proteins with the internalization signal provided by nucleic acid-binding domains (and nuclear localization signas) are autoantigens. This pattern is found with all allergens that I have examined. There are a few apparent exceptions, but in all of these cases, there is a closely related allergen from a related source that has the expected strong heparin-binding domain. It appears that in these cases, the less common allergen provides the initial exposure during the presentation phase of high inflammation, and the allergy is maintained by subsequent exposure to the more common allergen. After the establishment of the allergy, the strong heparin-binding domain is no longer needed, because antibodies bind to other parts of homologous allergenic proteins for internalization.

Just for fun, I have illustrated the honey bee allergen, hyaluronidase, to show both its strong heparin-binding domain (blue) along with its substrate hyaluronan (grey and red). Note that the substrate sugars are in the slot of the active site, which is lined with orange and yellow aromatic amino acids that provide flat, hydrophobic binding platforms for each sugar.

After this little distraction to provide further support for my explanation of the cause of allergies, I have to get back to looking at the role of leukotrienes in anaphylaxis, COPD, asthma and other inflammatory diseases.

Mast Cell Heparin

Mast cells are sentinels in tissues. They respond to invading pathogens by releasing their stored histamine, enzymes and heparin. The heparin modifies the activity of enzymes and cytokines.

What are mast cells and why are they loaded with heparin (left)? Mast cells start in the bone marrow, like many other components of the immune system. They then move into the blood stream and offload in most of the tissues that typically encounter pathogens and parasites. Thus, the typical commercial source of the mast cell-produced heparin is pig intestines or cow lungs, i.e. since heparin is made and stored in mast cells and mast cells are abundant in lungs and intestines, those are the sources of crude heparin. Proteins bound to the crude heparin are removed as the heparin is cleaned up to be used as an anti-clotting drug.

Mast cells are sentinels near the surface of mucus membranes that line the airways of the lungs and the digestive tract. Diseases of the lungs and intestines, e.g. asthma and inflammatory bowel disease, that have an inflammatory and/or autoimmune component yield high levels of mast cells in the affected tissues. Pathogens or parasites coming in contact with mast cells trigger the sudden release of vesicles full of histamine, enzymes and heparin.

Heparin stored in vesicles in mast cells can also be readily visualized by staining the mast cells in microscope sections using the fluorescent dye berberine (left). Berberine binds quite specifically to heparin and is also used in herbal medicine as a treatment for many inflammatory diseases, such as arthritis. It would be very interesting to know whether berberine has any effect on asthma.

Mast cells display a variety of receptor proteins on their surfaces. Protein receptors work by binding target molecules, ligands, changing their shapes and transmitting a signal through the cytoplasm. A key aspect of the signal transmission is the requirement for the ligand binding to bring together receptors in pairs. The pairing of receptors during ligand binding is facilitated by the binding of heparin to both ligands and receptors. Two ligands, e.g. cytokine peptides, such as TNF, can bind to adjacent sites on a heparin molecule and this pair can then bind to two receptors brought together on the surface of a cell. The receptors bind to the ligand and to the heparin. Some ligands will bind to their receptors without heparin, but the presence of heparin greatly accelerates and intensifies the reactions.

Heparin is synthesized in the vesicles of mast cells and binds to enzymes, e.g. tryptase, also present in the vesicles. The tryptase enzyme proteins form tetramers with heparin wrapped around the edge (left, edge view showing one pair of tryptase proteins with heparin bound diagonally to blue heparin-binding domains; other pair of tryptase proteins is hidden).

Interestingly the active site for each tryptase in the tetramer faces a hole where the four proteins come together. Thus the tetramer can degrade small peptides, but large proteins cannot get access to the blocked active sites. Monomers change shape and are no longer active.

Activated mast cells release their vesicle contents with some enzymes active and their bound heparin is replaced by the heparan sulfate attached to adjacent cells. Other enzymes are initially inactive bound to heparin and are activated by dissociation of the heparin once they are released from the vesicles. In both cases some of the heparin is released from the mast cells into the surrounding tissue. The free heparin can bind to cytokines released from other cells and the combined pairs of cytokines bound to heparin can in turn bind to appropriate receptors on other cells. The abundance of heparan sulfate bound to other cells will determine whether additional heparin is required for receptor responses from particular cytokines. Cells with abundant heparan sulfates will sweep heparin binding ligands toward receptors aggregated in lipid rafts, as the heparan sulfate proteoglycans are internalized for recycling.

Mast cells can be activated by allergens, because of IgE receptors. IgEs are antibodies that trigger allergic responses. The IgEs produced by antibody producing B lymphocytes circulate in the blood serum and bind to mast cell receptor proteins. Allergen molecules bind to the IgE-receptor complexes, trigger the activation of the mast cells and release histamine. The histamine binds to receptors on other cells and produces the symptoms of allergy or asthma.
Heparin can be sprayed into the lungs of asthma sufferers and reduce symptoms. This suggests that the ratio of heparin to cytokines is important and that cytokine signaling required for asthma episodes of airway constriction can bind individually to different heparin molecules and minimize mast cell triggering and histamine release.

Asthma also responds to a general decrease in chronic systemic inflammation. Thus, an anti-inflammatory diet and lifestyle, can reduce episodes and potentially reverse symptoms. Omega-3 oils and glucosamine, for example are both effective.

Tryptase model: Sommerhoff CP, Bode W, Pereira PJ, Stubbs MT, Stürzebecher J, Piechottka GP, Matschiner G, Bergner A. 1999. The structure of the human betaII-tryptase tetramer: fo(u)r better or worse. Proc Natl Acad Sci U S A 96(20):10984-91.


Berberine staining of mast cell heparin: Feyerabend TB, Hausser H, Tietz A, Blum C, Hellman L, Straus AH, Takahashi HK, Morgan ES, Dvorak AM, Fehling HJ, Rodewald HR. 2005. Loss of histochemical identity in mast cells lacking carboxypeptidase A. Mol Cell Biol. 25:6199-210.

Allergy, Asthma, Autoimmunity Start the Same Way

Inflammation is the current medical buzzword. Name the disease and inflammation is there.

Reproduction Requires Controlled Inflammation
Aspirin blocks many of the steps in triggering inflammation and thus, aspirin administration can be used to reveal a role of inflammation in many unexpected places. Aspirin is effective in blocking some forms of infertility, inhibiting miscarriages and ameliorating postpartum depression. So inflammation is a critical part of reproduction. But, also notice that depression is a symptom of chronic inflammation.

Cancer Requires Inflammation
High dose (IV) aspirin has been successfully used to treat cancer. Inflammation is required for cancer growth, because both use the same transcription factor, NFkB. The aberrant signaling of cancer cells would normally lead to programed cell death, apoptosis, but inflammation blocks apoptosis. Aspirin can in turn block NFkB and in the absence of inflammation, cancer cells die by apoptosis.

Inflammation is Self-Limiting
Aspirin also transforms the COX/lipoxidase system to produce anti-inflammatory prostaglandins/eicosinoids. Inflammation normally progresses into anti-inflammation. Blocking this progression leads to chronic inflammation and a shift from local to systemic inflammation with the rise of inflammatory interleukins in the blood stream.

Immune Response Requires Inflammation
The signal molecules (IL-1, IL-6, TNF) and transcription factor, NFkB, associated with inflammation were all initially identified in the development of lymphocytes. Hence, IL stands for interleukin, a hormone that triggers leukocyte (literally white blood cells or cells associated with the lymphatic immune system, i.e. lymphocytes) development. The nuclear factor, i.e. transcription factor, involved in expression of the large chain, kappa, of immunoglobulins in B cells, was called NFkB.

Genes Expressed by NFkB Cause Symptoms of Inflammation
About five dozen genes are under control of NFkB. Among these are COX-2, the enzyme that converts omega-6 arachidonic acid to inflammatory prostaglandins; iNOS, the enzyme that produces nitric oxide that dilates blood vessels to produce hot, red skin; and the inflammatory interleukins, IL-1, IL-6 and TNF, associated with autoimmune disease, fatigue and cachexia (wasting).

Autoimmunity and Allergy Start with Inflammation
Medical treatments focus on symptom abatement and ignore cause. What causes obesity, allergy or autoimmune disease? The answer appears to be chronic systemic inflammation plus exposure to unusual proteins. The unusual proteins are immunogenic, i.e. interact with the immune system to produce antibodies or reactive T-cell receptors, and are subsequently recognized as autoantigens or allergens, that are the targets for immune attack. Inspection of these autoantigens and allergens shows that they all have one thing in common, they bind to heparin via a strong heparin-binding protein domain that is typically a triplet of adjacent basic amino acids.

Heparin is a Short, Highly Sulfated Fragment of Heparan Sulfate
Commercial heparin is purified from the intestines of hogs and cattle. Heparin is released from mast cells (made fluorescent for microscopy using berberine) along with histamine and is released into the intestines to block pathogens from binding to the heparan sulfate that is part of the intestine surface. The heparin is anti-inflammatory and it contributes to minimizing the inflammatory response of the intestines to food.

Inflammation Reduces Heparan Sulfate Production
Pathogen-generated inflammation of the intestines reduces heparan sulfate production and increases immune response to food antigens. NFkB activation by inflammation turns off the production of some genes needed for heparan sulfate proteoglycan (HSPG) synthesis. Since HSPG is a major component of the basement membrane that holds tissues together, the reduction of HSPG results in protein loss (proteinuria) from kidneys, leaking of intestines, and disruption of the blood/brain barrier.

Reduction of HSPG Results in Immunological Presentation of Autoantigens/Allergens
Proteins are brought into cells by specific binding to protein receptors. In many cases, particularly involving signaling or growth factors, both the signal molecules and the receptors bind to heparin. In addition, there is a robust circulation of HSPG, which is secreted and internalized with a half-life of approximately six hours. The sweep of the HSPGs take heparin-binding proteins with them for internalization, e.g. HIV-TAT, heparanase, tissue transglutaminase. I think that this HSPG sweep under inflammatory conditions also internalizes basic autoantigens and allergens with strong heparin-binding domains. This internalization is the first step toward immunological presentation and the immune response to autoantigens and allergens.

Autoantigen/autoantibody/HSPG Complexes Kill Cells
Antibodies against self-antigens, autoantigens form antigen/antibody complexes that also bind to and cross-link HSPGs, because of the heparin-binding domains of the autoantigens. The large complexes may disrupt HSPG circulation and trigger apoptosis or abnormal physiology. There are many other examples of heparin-based complexes that are toxic, e.g. Alzheimer’s amyloid plaque, diabetic beta cell antibody complexes, celiac gluten/tRG antibody complexes, multiple sclerosis myelin antibody complexes, atherosclerotic plaque.

Anti-Inflammatory Diet and Lifestyle Protects
Dietary and lifestyle adjustments that minimize inflammation, e.g. low starch, no HFCS, low vegetable oil (except olive) and supplements of vitamins D & C, fish oil (omega-3) and glucosamine, reduce the risk of allergies/asthma, degenerative diseases and cancers. Simple, high level supplements with fish oil reduce numerous mental disorders, e.g. depression, ADHD; infertility, pre-eclampsia and postpartum depression; allergies, asthma; arthritis, atherosclerosis; burn recovery, septicemia and head injury.

Reducing Inflammation is a Panacea for Modern Diseases
Most modern diseases have an inflammatory component, because modern diets are rich in inflammatory components, e.g. starch/sugar, corn/soy oil, HFCS, trans fats, and exercise is minimal. The medical industry has not successfully promoted healthy eating and exercise; and in fact has promoted the devastating replacement of saturated fats with inflammatory polyunsaturated vegetable oils. Meat production has moved away from grazing on omega-3-rich plant vegetation to omega-6-rich corn and soy. Replacement of the corn/soy based agricultural economy would have predictably immense beneficial impact in reducing inflammation-based degenerative autoimmune diseases and cancers.

Topoisomerase Inhibitors

Inhibiting enzymes involved in DNA synthesis should stop cancer cells, because cancer is uncontrolled cell division. Topoisomerases are enzymes that help to relieve the twists on double helical DNA as it unwinds preparatory to replication. It appears logical that topoisomerase inhibitors should be cancer inhibitors. Unfortunately targeting DNA-binding proteins also targets most of the signal receptors that are the targets for the evolution of plant alkaloids.

Drugs are designed to be specific in their interactions with a particular target protein, but they are too small to be specific and end up binding to many other related proteins. Hence, drugs have side reactions that are to some extent unpredictable, because the interacting proteins are not known.

Aspirin, for example, is supposed to bind to and inhibit COX-2, the enzyme that converts omega-3 and omega-6, long-chain fatty acids into corresponding anti-inflammatory and inflammatory prostaglandins, resp. Aspirin also binds to proteins that inhibit NFkB, the transcription factor that controls expression of inflammatory genes. Aspirin binds to dozens of other proteins. Aspirin does lots of other things than just blunt inflammation, but those side reactions are usually not significant enough to get our attention.

Heparin is one of the most commonly used drugs. It binds to and activates an inhibitor of thrombin, an enzyme that activates fibrin and mediates clotting. Heparin also binds to other components of the clotting system, as well as a dozen components of the complement system, and most of the cytokines that control communications throughout the body. When patients are given heparin injections, heparin binds continually to all of these components and must be constantly supplemented and monitored. Inflammation depletes the heparin components throughout the body, so it is not known prior to injection, how much heparin will be needed to saturate other serum proteins before the desired level of clotting inhibition is achieved. This illustrates rather dramatically that most drugs have only limited specificity.

One of my students provided another example of the minimal specificity of small molecules, especially the alkaloids and phenolics produced by plants. He brought to me a research article espousing the use of phenolics from yerba mate, which serves as a coffee-like stimulant in Argentina, as a topoisomerase inhibitor and potential anti-tumor treatment. Sure enough, phenolics extracted from this plant inhibit topoisomerase, and they may well be able to inhibit the growth of tumors, but it is doubtful that the binding of the phenolics to topoisomerase in the tumor nuclei has anything to do with inhibition of tumor growth.

Topoisomerase binds to nuclear DNA as the DNA unwinds during replication to produce two new double helical DNA molecules. Topoisomerase is a DNA-binding protein, i.e. a protein that binds to a negatively charged polymer of small deoxyribose sugars and flat purine and pyrimidine bases. Proteins bind to DNA in two ways. Amino acids of the protein either bind along the edges of the hydrophobic stack of base pairs, e.g. sequence-specific transcription factors, or they provide hydrophobic, flat surfaces that bind to the hydrophobic faces of the separated bases. Topoisomerase does both, because it deals with single-stranded regions of DNA and therefore binds to both the phosphates, as well as the bases. The important point here is that both aromatic amino acids, with flat hydrophobic rings, and the hydrophobic tails of basic amino acids, i.e. lysine and arginine, bind to the hydrophobic faces of nucleic acid bases.

I have illustrated the binding of a “topoisomerase inhibitor” to show the arginine (blue) in the active site cleft of the topoisomerase that binds across the hydrophobic face of the inhibitor (grey and red). Many plant phenolics and alkaloids would be expected to similarly bind and act as inhibitors of topoisomerase. This observation and the ease by which alkaloids enter cells (attached to circulating heparan sulfate?) suggests that a major function of the nuclear envelope may be to minimize access of alkaloid and related molecules to the nucleic acid binding proteins of the nucleus.

The binding promiscuity of secondary plant products is further exemplified by berberine. Berberine is an alkaloid found in goldenseal and is an herbal remedy used to treat a variety of inflammatory diseases. It also binds to heparin (and nucleic acids) to produce a fluorescent complex. Thus, mast cells that store and secrete histamine and heparin to produce the symptoms of allergy, can be vividly stained with berberine.

I could not resist the temptation to check to see if berberine also binds to topoisomerase. A quick search of the research literature showed that berberine is in fact a topoisomerase inhibitor.

The numerous cross reactions of drugs are further illustrated by metformin, the common drug used in the treatment of type II diabetes. Metformin is approximately planar and provides a surface that cannot hydrogen bond, i.e. it is hydrophobic. I expected that metformin would bind to tryptophans that I observed as common substrate-binding amino acids in the active sites of proteins that bound to polysaccharides, e.g. lectins, glycosidases and glycanases. To test this, I had students in one of my courses examine the inhibitory activity of metformin on E. coli beta-galactosidase. They found measurable inhibition and support for competitive binding to the active site that contains a pair of the predicted tryptophans.

My protein modeling and structural studies show the basis for numerous interactions between plant secondary compounds, drugs, nucleic acids, polysaccharides (glycosaminoglycans, e.g. heparin) and proteins. Unpredicted cross reactions abound and every drug can be expected to interact with multiple proteins. This provides a note of caution to the use of any drug and encourages minimal exposure, since many unobserved and unanticipated side effects are occurring. These observations also question routine ingestion of herbal remedies, after all, plants use their secondary products as potent defenses against being eaten. Alkaloids disrupt nervous systems and cellular signaling. Plants are not naturally safe.

Rattlesnake Venom

Plants attack pathogens, pests and herbivores with toxic chemicals, whereas snakes attack other animals with heparin-binding protein toxins. The rattlesnake toxin, crotamine is an example of a small peptide cross-linked by disulfide bonds, which attach to cells via heparin-binding domains.

I had a sabbatical in Singapore, at the National University. I was seated on the patio of the university canteen eating one of my typical lunches: curried mutton with hot lentils, rice and a durian milkshake, served on a banana leaf. I struck up a conversation with a biochemist who studied the structure and function of snake venom proteins. He was systematically analyzing the proteins in various venoms looking for proteins that interact with essential features of cells. This was a potential initial step for the design of new drugs. I remember asking him how he knew which part of the venom proteins was important and which parts just served as a rigid platform to display the active parts. He said that it was simple, the water-binding, hydrophilic amino acids that formed amorphous loops bordered by sulfhydryl-bonding cysteines were his targets. Ten years layer, I observed that these loops also have the basic amino acids (K, lysine and R, arginine) that form heparin-binding domains.

His conversation came back to me a couple of days ago when I ran across the structure and function of the crotamine toxin from the venom of the South American Rattlesnake, Crotalus durissus terrificus.

YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWKCCKKGSG

The heparin-binding domains were evident in both the amino acid sequence as well as the protein structure. As is true of many small proteins, or peptides, they are held in their functional shape by -SS-, disulfide bonds, between cysteines (C). The presence of two well-defined heparin-binding domains (blue) also predicts that the toxin would be anti-bacterial and that it would bind to phospholipids, i.e. membranes. The observed toxic quality of the toxin is its ability to disrupt ion transport through membranes and it has a shape similar to the mammalian anti-bacterial peptides, defensins. Most venom toxins bind to the heparan sulfate proteoglycans (HSPGs) of their victim's cells and then as the HSPGs are brought close to the cell surface during recycling, the toxins attack the membrane proteins and kill the cells.

I am tangentially interested in snake venom, because the proteins that mediate its toxic effects are related to the hormones that mediate inflammation.

It is interesting that a simple discussion over curried mutton ten years ago would be so consistent with a major shift in my research interests to study inflammation.

Preventing Allergies

The cause of allergies and autoimmune degenerative diseases is inflammation.

As a scientist, I am concerned with how the body works at the molecular level. I try to understand how molecules of cells interact to cause disease. So if you tell me that you have an allergy, I want to understand how you became allergic and I am much less interested in how you avoid triggering your allergy. If you say that your allergy is triggered by ragweed pollen, I want to know the shape and structure of the proteins or carbohydrates of the pollen that actually come in contact with receptors on the surface of your cells and trigger the allergic response, but I also want to trace those interactions back to the original events that started the allergy.

Allergies are mistakes of your immune system. Your body should learn to ignore common food and environmental molecules as it ignores itself. There is an elaborate system used by cells of your body to disassemble and display fragments of dangerous pathogens on the surface of cells for evaluation by the immune system. Inappropriate display of innocuous or self molecules is part of the problem in allergies and autoimmunity.

Ragweed pollen, for example, will cause no reaction unless pollen proteins bind to antibodies (IgE) held in receptors on the surface of mast cells. We know that ragweed pollen binds to anti-ragweed antibodies on the surface of mast cells of allergic individuals and triggers the release of histamine and other molecules that give the symptoms of allergy. That’s why you take antihistamines to remediate the allergic symptoms. The first questions are what are the ragweed molecules to which the antibodies bind, i.e. the ragweed allergens and why is this allergic person producing anti-ragweed antibodies?

We know that the ragweed allergens are common pollen proteins, but why are they particularly prone to producing allergies? I tried to figure out this riddle by asking if there is something about these proteins that make their transport into cells more likely. I had just discovered that a particular amino acid sequence, a triplet of basic amino acids, lysine or arginine, resulted in transport of proteins into cells. This triplet that provides binding to heparin is found, for example in the nasty HIV protein, called TAT that moves on heparan sulfate proteoglycans (HSPGs) from infected to uninfected human cells and paves the way for the spread of infection. This triplet is also found in heparanase, that is first secreted by cells in an inactive form, is brought back into cells by binding to HSPGs, is activated by partial digestion and resecreted for action in the extracellular environment. This heparin-binding triplet can also be added to other proteins, e.g. the fluorescent jellyfish protein, to transport those proteins into cells.

Examination of ragweed pollen and subsequently dozens of other common allergens revealed that each one (or a close relative) possessed the unusual heparin-binding triplet of basic amino acids. The basic charged character of these sequences also determined that these parts of proteins would be present as accessible coils on the surface of the proteins. It is interesting that people suffering from the autoimmune disease of lupus produce antibodies to most of the proteins found in the nucleus of their cells. These nuclear proteins bind to nucleic acids, that mimic the structure of heparin and in many cases have triplets of heparin-binding basic amino acids. Thus, it appears that allergenic proteins enhance the chance of uptake by cells that can display them to the immune system, because of their triplet heparin-binding domains and the immune system subsequently produces antibodies that bind to other regions of the protein allergens. This explains how the antibodies are produced to these allergens, but it does not explain why some people produce antibodies to environmental antigens and healthy people do not.

Allergic people readily expand their allergies to include new allergens. What is it about these susceptible people that makes them allergic? I think that the answer is inflammation. Inflammation leads to a disruption of normal production of heparan sulfate proteoglycans and as a consequence to a change in how external proteins interact with cells involved in processing antigens for presentation to the immune system. This means that people with chronic inflammation, also called the metabolic syndrome, are not only increasingly susceptible to diabetes, arthritis, heart disease, etc. but they are also at risk for picking up new allergies. This also suggests that an anti-inflammatory diet and lifestyle changes would be of great benefit to those with allergies. Unfortunately, because of immunological memory, it will take years to deplete the population of antibody secreting cells that provide the basis for a specific allergy and during this depletion time, the allergen would have to be scrupulously avoided. It might also mean that autoimmune diseases such as type I diabetes might be treated by depletion of anti-beta cell antibodies and their secreting B cells along with a shift to an aggressively anti-inflammatory diet.

It is my belief that many of the genetic components of allergies and autoimmune diseases would not be experienced in the absence of chronic inflammation as a precipitating condition.