Health and Heparan Sulfate Circulation — Connective Tissue is Alive

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Arthritis, Alzheimer’s, diabetes, cardiovascular disease, osteoporosis, cancer, etc. are all diseases of cellular metabolism and secretion.  What goes on inside cells and on their surfaces explains a lot about health and why we get sick.  Cells feed off of what’s around them, use some of those materials to replicate and package up cell-made materials for export.  Eat, replicate and secrete.  Symptoms of disease result if those processes are compromised.

Cell that make Cartilage, Eat Cartilage

The connective tissue that makes up the cartilage of tendons and the non-mineral parts of bones, as well as a layers of skin, is made up of proteins (collagen) and polysaccharides (glycosaminoglycans, GAGs), e.g. heparan sulfate, hyaluronan and chondroitin sulfate, produced by  chondrocytes or fibroblasts.  These proteins and polysaccharides are synthesized and then secreted by cells.  This process goes on continuously, since the connective tissue is alive and literally crawling with cells that make the cartilage.  To keep the connective tissue healthy, the old tissue has to be digested, so that new material can replace it.  Thus, the cells that live in cartilage also eat cartilage.  These cells get all of their nutrients, e.g. protein and carbs, from eating cartilage.  They don’t get glucose and amino acids, or even oxygen (they ferment), from the blood, because there are no blood vessels in cartilage.  The photomicrograph at left shows the red chondrocytes surrounded by a light capsule of heparan sulfate as they burrow through the purple cartilage.  The next micrograph shows the cytoskeleton of actin filaments (stained with a red fluorescent dye, that lies under the cytoplasm of a chondrocyte.  Motor proteins move other proteins, such as syndecans, the proteins to which the heparan sulfate chains are attached, through the  cell membrane (see the animations below.)  The last micrograph shows the green stained microtubule network on which vesicles move to carry heparan sulfate products from one end of the cell to the other (under the actin and past the orange-dyed nucleus) during synthesis and digestion.

Chondrocytes Burrow Through Cartilage

Chondrocytes are the cells that eat and make cartilage, but all of this eating and making goes on at the same time that the cartilage is also holding everything together, i.e. it is still strong.  If cartilage is cut and the cut ends are held tightly together, the chondrocytes will knit the cartilage together and it will become as strong as it was. 

Heparan Sulfate Circulates over the Surface of Cells

Chondrocytes are not actually rigidly embedded in the cartilage, but rather maintain a capsule of heparan sulfate around themselves.  Thus, they continue to secrete a mixture of heparan sulfate, chondroitin sulfate and collagen, but the heparan sulfate is recycled through the capsule and the other molecules merge into the existing cartilage.  Thus, the heparan sulfate is a kind of carrier that keeps the cartilage from “setting up” while it is being made and transported.  Other cells of the body, such as neurons, don’t make cartilage, but they still have heparan sulfate (HS) circulation that is intimately involved in many other processes, such as the action of hormones.  Disruption of HS circulation causes the symptoms of Alzheimer’s or type 1 diabetes, for example, since amyloids assemble as filaments on threads of HS, and the amyloid filaments jam essential HS circulation.  Plaque in atherosclerotic vessels is high in HS content.  HS is also a major component surrounding vessels to form the blood brain barrier and the barrier to protein loss from kidneys into urine or loss into the gut lumin.  Heparin (fragments of HS) is continually released from mast cells in the lining of the gut to prevent pathogens from binding to cell HSPGs. 

HS Sweep the Cell Surface

There is a constant flow of heparan sulfate proteoglycans (HSPGs) through the cell membrane from the rear of the chondrocyte to the front where the HS is digested again and the protein that was embedded in the membrane, syndecan, is recycled to the Golgi for another trip.  HSPGs (animation to left with blue protein and yellow HS) are attached to motor proteins that propel them through the membrane along microfilaments of actin that form the cyctoskeleton just under the membrane in the cortical region of the cell.  Thus, the heparan sulfate of the HSPGs stick out like hair from the cell surface and sweep continuously from the back to the front of the cell.  At the front of the cell, the HS sweeps through the intact cartilage and reverses the process of cartilage assembly.  The chondroitin sulfate, collagen and HSPGs are dragged into the cell and digested.  The protein parts of the HSPGs are transported to the Golgi  and the HS is synthesized along with other cartilage components and moved in vesicles along microtubules before it is secreted.

HS is Secreted at One End and Eaten at the Other

The animation left shows 1) the initial digestion of the cartilage proteins and polysaccharides on the left.  These cartilage components of amino acids and sugars, are used by the chondrocytes as their sole nutrients 2), and to produce new proteoglycans 3) HS and chondroitin sulfate proteoglycans, in the Golgi, are 4) packaged into secretory vesicles and are 5) secreted on the right.  The HS chains, attached to proteins, are 6) swept through the membrane (see the first animation above) toward the front of the cell, leaving the collagen and chondroitin sulfate for form cartilage behind.  In the process, the heparan sulfate proteoglycans 7) disrupt and solublilize old cartilage ahead as the chondrocytes 8) move through the connective tissue like moles digging through soil.

Other Cell Processes Involving Heparan Sulfate:

• Amyloids of Alzheimer’s and type I diabetes assemble bound to HS.
• Hormones bind to receptors wrapped around HS.
• Blood clotting is controlled by HS.
• Complement is controlled by HS.
• Blood brain barrier is composed of HS.
• Kidney protein barrier is composed of HS.
• Inflammation blocks HS synthesis and promotes heparanase synthesis.
• GAGs are animal soluble fiber when eaten and feed gut flora.
• Pathogens bind to HS.
• HIV-TAT is transported between cells by HS circulation.
• Heparin is made by heparanase fragmentation of HSPG in mast cells and is secreted along with histamine. 
• NFkB activation inhibits HSPG production and stimulates heparanase production.
• Heparan sulfate proteoglycans organize nerve synapses and acetylcholine esterase binds to HS. 
• Gastric proteases cleave around heparin binding domains of proteins, e.g. milk, consist of clusters of basic amino acids.  Peptides with heparin binding domain are antimicrobial; all of the heparin binding peptides are subsequently degraded by pancreatic proteases.
• Heparanase is initially secreted inactive and bound to HSPGs, but it remains bound and is internalized again along with the recycling HSPGs, and is activated before being secreted again.
• Allergens and autoantigens are unusual proteins with sequences of three adjacent basic amino acids (arginine or lysine) that require HSPG circulation for presentation of the immune system.  Nuclear proteins that interact with nucleic acids have sequences of four basic amino acids, the nuclear translocation signal, and are therefore common antinuclear auto antigens.

Health Diagrams

Food Poisoning and Manmade E. coli

Bacteria on food is a problem for diet-compromised people.   

Gut Flora are Required for a Healthy Immune System

Healthy people don't get sick from food poisoning, because their gut flora provide protection.  Gut bacteria control the development of the human immune system by producing interesting compounds, including short chain fatty acids and vitamins.  In response to the gut bacteria, the healthy immune system produces white blood cells that can effectively attack bacteria, and also control this aggressive behavior to spare human cells and avoid unnecessary attacks on beneficial bacteria.

Disrupted Gut Flora Lead to Susceptibility to Disease/Infection

Gut flora can be compromised by what we eat and antibiotics.  Those normally affected by food poisoning are the very young (on formula), the old (constipated) and those treated with antibiotics.  Each of these groups have abnormal gut flora.  Food poisoning is rarely observed in exclusively breastfed babies being introduced to foods, because human milk contains potent antimicrobial polysaccharides (human milk oligosaccharides) that only permit the growth of a few species of Bifidobacteria.  Formula (in any amount) disrupts the normal development of the gut and immune system by stimulating an inflammatory growth of adult gut bacteria, making these babies more susceptible to intestinal and respiratory diseases, including food poisoning.

Constipation, which is more common in older people, reflects a disruption of the gut flora and decreases the effectiveness of the immune system in these individuals.  In most cases the compromised gut flora results from a long history of a restricted diet and  reduced access to environmental sources of bacteria.

Antibiotics are usually ignored as major corruptors of the immune system, even though they are known to produce diarrhea and constipation.  Doctors reluctantly suggest that people taking antibiotics should just eat some yogurt.  This is a silly oversight that severely compromises future health, because probiotics supply only a tiny fraction of the 150 different species of bacteria needed for a healthy body and immune system.

Pathogenic E. coli is Made by Antibiotic Use in Cattle

E. coli is a common and essential resident of the human gut and the best studied bacterium.  This bacterium is not normally resistant to antibiotics nor does it produce deadly toxins.  Antibiotic resistance and toxin production results from treating cattle with antibiotics to increase fat production prior to butchering.

Antibiotics Select for E. coli that Stick to Rectal Surface of Cattle

Pathogenic E. coli are not found throughout cattle fecal material, but rather they are only in the outermost surface layer.  This outer layer of material contains bacteria from the surface of the rectum just as the cow pies are deposited.  E. coli does not normally stick to this surface, because it lacks a protein, such as a hemagglutinin capable of binding to the surface polysaccharides, heparan sulfate.  Antibiotics kill off the bacteria normally residing on the surface.  As a member of the intestinal biofilm community, E. coli continually exchanges DNA/genes with other bacteria in the gut and picks up three useful genes, to become a pathogen:

1. Antibiotic resistance
2. Hemagglutinin for sticking to surfaces
3. Toxin to release nutrients from the intestinal walls.

E. coli with these three genes can colonize the rectal tissue of cattle in feed lots.

Pathogenic E. coli Can be Easily Avoided

We have to work hard as a society to have problems with E. coli.  Pathogenic E. coli results from absurd use of huge quantities of antibiotics just to disrupt the normal gut flora of cattle so that they become unhealthy and store fat in their tissues, i.e. prime beef.  The same effect can also be achieved just by feeding the cattle some short chain fatty acids, or better still avoiding this step by feeding exclusively on grass.  It would also be easy to treat the few cattle that have pathogenic E. coli, so that it doesn't become a problem.  Proper treatment of manure and meat processing would also block transmission of pathogenic E. coli to agricultural crops or meat.  Finally, an Anti-inflammatory Diet and Lifestyle would provide a healthy gut flora and immune system that would make people less susceptible to the pathogen.

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.