Extracellular Macromolecules Glycosaminoglycans; proteoglycans; glycoproteins; mucins Glycoprotein synthesis; plasma proteins Molecular immunology: innate immunity; inflammation Molecular immunology: adaptive (acquired) immunity Fibrous proteins: keratin, collagen and elastin

Extracellular Macromolecules Compositions are combinations of carbs and protein. Lipids have a non existent role here. Combinations of polysaccharides and proteins. Today, these substances known as glycosaminoglycans, glycan is a synonym for polysaccharide. Next macromolecules that are predominantly proteins, proteoglycans, then more important in functional sense, two lectures on molecular immunology, two areas, immune system that is innate, and we will start to look at some of the molecular aspects of inflammation. Process that governs infections, interactions with microbial world. Then process where by antibodies are made, acquired immune system is another topic, then extracellular macromolecules fibrous proteins, such as collagen. First structures, tie them to properties. 1. Glycosaminoglycans Proteoglycans Glycoproteins Mucins

Extracellular Macromolecules macromolecule % carb. glycosaminoglycans* (GAGs) 100 proteoglycans* 90-95 glycoproteins 2-30 fibrous proteins 1-2 Examples of functions: mechanical support lubrication cushioning adhesives cell spacers selective filters Here are the extracellular macromolecules classified in structural terms, by how much carb or protein they have. As we go down the table, start out with substances that are only carbs, polysaccharides then substances that are predominantly carbohydrates. Then third line, glycoproteins, mostly proteins that have some sugar as part of their structure. As you see fibrous proteins fall into this category, only in that they have token amount of carbohydrate, essentially protein, polypeptide. Then, short list of types of function these substances are involved in. These are here to indicate the diversity of function that these are involved in. These two terms, glycosaminoglycan, often abbreviated as GAG’s, can see that glycan, polysaccharide, and its modified by the fact that the sugar in these glycans has a nitrogen and amino group and that distinguishes them from the typical polysaccharide such as cellulose and glycogen. These go by other names, mucopolysaccharides, heard about those in histo. Then the proteoglycans also go by mucoproteins. Muco meaning that both substances were first found in mucous secretion, characterized from those. That’s why these terms are considered obsolete because they imply that they are confined to mucous secretion, where in fact they are just about everywhere. * aka mucopolysaccharides, mucoproteins, respectively 1

Extracellular matrix in tissues ground substance + fibers macromolecules between cells ground substance molecules GAGs/proteoglycans (mostly carbohydrate) fibers fibrous proteins: structural adhesive especially abundant in connective tissue Where these substances fit in. the extracellular matrix is composed of ground substance, older term, material that is more or less fibrous. These names came about from macroscopic approach. Have these large molecules that exist between cells consisting of ground substance molecules, now know as glycosaminoglycans and combos of that and proteoglycans. Fibrous materials, primarily fibrous proteins that play roles that are structural, support the surroundings, also important adhesive properties, help to hold the structures and materials together. In general these are materials more in connective tissue. Picture shows epithelial surface that has only the cells, further down, have the layers consisting of extracellular matrix. epithelial cells adhesion molecules extra- cellular matrix basal lamina underlying cells 2 Adapted from Hypercell

GAG structure exist as: A sugar exist as: independent molecules e.g., hyaluronate & heparin parts of larger structures e.g., in proteoglycans heteropolysaccharides repeating structure: disaccharide (AB)n ABABAB… where A is usually 1 uronic acid (hexose with C6 as COO– ) & B is 1 glycosamine (amino sugar) derivative unbranched glycosidic linkage anomeric C of 1 unit linked to hydroxyl of adjacent unit B sugar Chemical structures of these materials, GAGs have a structure consisting of, polysaccharides, and like all polysacc the units that oppose the polysacc are monosacc linked up in characteristic way. The GAG’s have an independent existence, the molecules are polymers on their own and the two ones we will talk about aout are hyaluronate and heparin, GAG’s of 100 percent polysacc. Also the GAGs are parts of other structures ,the proteoglycans. The GAG structure consists of these two types of sugars, recognize that hexose similar to glucose, except that there is a modified part of this structure, instead of being glucose, which has a hydroxyl group on numebr 6, it’s a carboxyl group. Known as uronic acid. We have glucuronic acid, sugar derived from glucose, and the polymer structure consists of , heterosacc, not every unit is identical. So far, glycogen cellulose and amylose, its glucose over and over, those are homopolysacc. Here its heterosacc, repetition is of disaccharide. If we have the A sugar, and the B sugar, the polymer would be some variation on repeating the A unit linked to the B unit, that’s what makes these polysaccharides. Abbreviate where AB is disaccharide repeated n times. Here is the A sugar, uronic acid, a carboxyl group, and the B sugar is an amino sugar, we have the prototype is glucose but one hydroxyl, number two is now amino group instead of hydroxyl group, glycosamine in general, or if its glucose then glucosamine. These polymers are also unbranched, linear chains, and linkage, these are the three sugars, linkage involves anomeric carbon, number 1 carbon generally, only one that has two bonds to a heteroatom, usually oxygen. And that is known as glycosidic linkage. The linkage then goes to a hydroxyl of adjacent one. 3

GAG structure: repeating units 4 GAG A sugar B sugar hyaluronate glucuronate N-acetyl glucosamine * 2 5 Glycosidic linkage between A sugar and a B sugar where the linkages are B 1,3, sometimes alpha sometimes beta, always involve number one carbon and then number 3 or 4. this other linkage is beta 1,4. polymer, GAG is hyaluronate, the A sugar is glucuronate and the B sugar is glucosamine where amino group is acetylated.

GAG structure: repeating units 4 GAG A sugar B sugar hyaluronate glucuronate N-acetyl glucosamine chondroitin sulfate glucuronate N-Ac galactosamine 4-SO4 dermatan sulfate iduronate " heparan sulfate glucuronate glucosamine N-SO3, 6-SO4 heparin iduronate 2-SO4 " keratan sulfate galactose N-Ac glucosamine 6-SO4 *opposite configuration in iduronate glucuronate/iduronate: epimers at C5 glucose/galactose: epimers at C4 * 2 5 Others where GAG has these names, with chondroitin sulfate the A sugar is glucuronate, but the B sugar is an amino sugar but instead of glucosamine its galactosamine. The difference between glucose and galactose, epimers of one another, number four carbon, put a line that represents the way the hydroxyl group comes off. Minor difference. The other difference, chondroitin has instead of acetyl group, the hydroxyl group is a sulfate. This hydroxyl in number four position in a different direction and sulfated instead of being free hydroxyl group. Another variation is dermatan sulfate. Initially isolated from skin. A sugar is iduronate, difference is config around a single carbon, here its the number 6 carbon, line represents the config of carboxyl, so it comes off down instead of up. Main point is there are structural differences and they are seemingly minor. In some way these small structural diff translate into significant difference in function. Then heparin sulfate, glucuronate is the A sugar, amino sugar is sulfated in tow places, hydroxyl and n sulfate in number 2 position. Heparin is iduronate, sulfated, then keratan sulfate is where A sugar is not a uranic acid, actually galactose. This gives at least some idea of the range of structural variation among these structures. Again in this column they are GAG’s that have these variations of this repeating structure.

GAG structure: repeating units GAG A sugar B sugar hyaluronate glucuronate N-acetyl glucosamine chondroitin sulfate glucuronate N-Ac galactosamine 4-SO4 heparan sulfate " glucosamine N-SO3 6-SO4 heparin iduronate 2-SO4 " dermatan sulfate iduronate N-Ac galactosamine 4-SO4 keratan sulfate galactose N-Ac glucosamine 6-SO4 *opposite configuration in iduronate * 2 5

Hyaluronate (aka hyaluronan) 5 mol wt: 106 – 107 (5000 – 50,000 monosaccharide units) very polar: 2 hydroxyls/unit 6 heteroatoms/unit COO– every other unit binds cations: Na+, Ca++ Display of HA in motion A B A B A B Huge molecule, at least a million molecular weight. Important property, knowing the hydroxyl groups are there, that this is a polar structure, each unit has two hydroxyl groups and every other one has a charged group, carboxyl group. This will be a polar molecule, poly anion, will bind positively charged things like sodium and calcium, any cationic substance that is out in the extracellular space. As a result of the negative charges, any like charges repel one another, makes it an extended structure. hyastk2.gif – – – 1 2 3 4 5 6 (glucuronate–N-acetyl glucosamine)3 (glcUA–glcNAc)3

Hyaluronate: structure & properties 6 extended structure (charge repulsion) hydrophilic: binds 10 –100 × wt in H2O additional, loosely associated H2O, so that volume occupied ~1000 × weight Display of HA with glcUAs in CPK Negative charges are spaced maximally, that makes this structure very extended. This is six unit here. Extended structure is a consequence of its high density of negatively charged carboxyl groups. hyacpk2.gif – – – 2 3 1 4 5 6 (glcUA–glcNAc)3 glcUAs in space-filling form (CPK)

Hyaluronate solutions viscous, gel–like, compression-resistant occurrence: EC matrix, esp. in developing tissue healing wounds synovial fluid functions: lubricant shock absorber flexible cement attachment site path for cell migration made by fibroblasts degraded by hyaluronidase hyaluronidase bacterial hyaluronidase facilitates spread of infection Alberts et al. Fig. 19-37 Thousands of these repeated units. Solutions that contain hyaluronate are viscous compared to water itself. They are more akin to a gel like structure. One property is it makes the material more resistant to compression, distortion and change. Find these in various parts of extracellular matrix, tissue that is developing where there is a lot of cell turnover, one of the ways that space is made is this hyaluronate. Wounds that are healing have high concentrations of this. Over time fluid in many joints, synovial fluid, has its properties of something similar to egg white, gelatinous. Glycogen particle, looks like this, if we look at fibrous protein. One molec of hyaluronate enormous volume, at this size or this level, does look like this structure, is not extended, at this level of magnification, this stretch would be a huge number of monomer units. Eventually it does bend slightly and with perhaps fill a relatively compact space. Here are some of the functions that have been attributed to hyaluronate, fluid containing it would act as lubricant. By resisting compression and changes of shape, property as shock absorber, at higher concentration acts as cement, adhesive material that is not a rigid cement but flexible. These molecules are places where cells will attach and by providing space that don’t contain cells, means by which cells can migrate during process of development. Synthesized by the cells known as fibroblasts. They undergo turnover, enzyme that breaks down these macromolecules known as hyaluronidase. Significant fact is that some bacteria secret such enzymes, which when they infect the ECM are important in breaking down the material that is in that space, enables the bacteria to spread. Not all bacteria have this, typically in infection that is more serious, going to because host defenses don’t thwart the tendency for bact to spread out. 7

Heparin mol wt ~ 104 ~ 40 monosaccharide units made & released from mast cells in lungs & liver GAG diff because of size, small molecule. Fewer monosacc. Here is the repeating structure, each monomer has two negative charges, so this would be a extended and polyanionic material. In the resting state, heparin is on the surface of the cell. Heparin goes into action when something cleaves the heparin from cells surface, now its free to do its job. heparin cell 8

Heparin mol wt ~ 104 ~ 40 monosaccharide units made & released from mast cells in lungs & liver also associated with luminal surface of endothelium anticoagulant forms complex with antithrombin III this complex binds to thrombin, inactivating it as a result, clot growth is limited fast-acting, making it therapeutically useful Short description is anticoagulant, interrupts and slows process of blood clot. Here is the means by which it does that, something that is used therapeutically, anticoagulating that can be injected and halt clotting. Heparin has the value that it is very fast acting, anticoagulant. heparin cell 8

Extracellular Macromolecules macromolecule % carb. glycosaminoglycans* (GAGs) 100 proteoglycans* 90-95 glycoproteins 2-30 fibrous proteins 1-2 Examples of functions: mechanical support lubrication cushioning adhesives cell spacers selective filters The next category are proteoglycans, that name implies that these are substances that are polysacc. Also contain protein, 5-10%. * aka mucopolysaccharides, mucoproteins, respectively

Proteoglycans (PGs) 9 Examples composed of as many as 200 GAG chains covalently bonded to a core protein via serine side chains molecular weight range: 105 – 107 GAG chains: chondroitin sulfate, heparan sulfate, dermatan sulfate, keratan sulfate Examples decorin many connective tissues binds type I collagen, TGF-β perlecan basal laminae structural & filtering function aggrecan syndecan (slide 13) from Alberts et al. Fig. 19-57 The proteoglycans or PG’s contain a variety structurally, always have a GAG and they always have at least one protein. Proteoglycan example of PG, protein in green, generally known as core protein, and GAG is dashed line in red. Many have more than one GAG chain but this one, decorin is the simplest example. In general, GAG chains are linked covalently to the protein and by way of serine side chain, hydroxyl side chain, and they have this wide rage of molecular weight. GAG’s mentioned, these sulfates, these are the names of the GAG parts of these structures. Decorin is found in many connective tissues, often bound to one of the collagen types. Perlecan has different arrangement of core proteins and several GAG chains attached, and then aggregan ,extended core protein, with a very large number of GAG chains attached in this fashion , bottle brush analogy. Alberts T 19-3: Dcrn GAG chndSO4 /drmSO4 GAG chains core protein 9

PG in basal lamina of renal glomerulus adapted from Alberts et al., 3 ed., Fig. 19-56 network of fibrous proteins & perlecan PG forms filter Here is a surface, made up of a few types of proteins, collagen, fibrous protein. Here is proteoglycan that forms part of this structure. Meshwork functions to act as a filter. This part of the kidney in effect filters out big things from little things, little things like water and sodium chloride get through ,but cells and proteins, that are part of blood, if this is filtering blood are held back by meshwork, and they don’t bind to this structure, delicate process. This is an example of how a complex molecular structure is composed of some of these PG’s and fibrous proteins. entactin GAG: heparan SO4 perlecan laminin type IV collagen 10

Proteoglycans: aggrecan ~100 GAG chains/molecule ~100 monosaccharides/GAG chain each "bristle" = 1 GAG chain each GAG chain is either chondroitin sulfate or keratan sulfate GAG chains linked to ser side chains of core protein The aggrecan, single core protein linked up to a few GAG chains that can be either chondroitin sulfate or keratan sulfate. based on Alberts et al. Fig. 19-37 4ed. 19-40 core protein GAG chains 11

An aggregate of aggrecans & hyaluronan ç 1μm è major GAG–PG in cartilage link proteins bind noncovalently with bound H2O, disperses shocks, compressive force ~ cell size adhesion proteins link to collagen & cells degraded by chondroitin sulfatase, etc core protein These get more complex, structure seen in cartilage commonly, consisting of several aggregans, presented by bottle brush structures that are non covalently bound by link proteins to a single large molecule of hyaluronate. That’s what the blue line represents, result of this aggregate is that the material is very good at resisting compressive forces and its huge, roughly the size of a cell. This represents the whole structure of which this picture is just a small part. A few microns in length. These things can get huge, make the point that there are enzymes that break these materials down, and some times there are bacteria that can secrete these enzymes, makes these bacteria bad guys in the sense that they will contribute to destruction of tissues over time. In gingivitis, that goes on to perio disease, this is part of the story, these materials, enzymes that contribute to break down and irreversible breakdown of tissue. link proteins hyalur- onan keratan sulfate chondroitin sulfate 12 Alberts et al. Fig. 19-41

Proteoglycans: syndecan cell-surface PG core protein domains intracellular transmembrane extracellular 5 GAGs attached functions interactions cell-cell cell-matrix growth factor receptor GAG chains Even though they are primarily ECM, core protein extends inside the cell. Trans membrane domain, and outer part has the GAG chain. This syndecan and PGs like it, important in interaction between cells and cells in extracellular matrix. outside inside core protein Lehninger et al. Fig. 9-22 13

GAG synthesis & breakdown activated precursors: UDP–monosaccharide derivatives e.g., UDP–glucuronate residues added one at a time in Golgi complex sulfate moieties donor: PAPS (active sulfate) degradation lysosomes specific glycosidases & sulfatases mucopolysaccharidoses genetic disorders accumulation of GAG due to absence of a specific glycosidase or sulfatase –UDP There is the way that they are synthesized, activated precursors are the story as with all polymers. Activated precursor for sugar is sugar linked up to UDP. UDP glucose is activated precursor for glycogen. Sulfate comes from sulfate donor known as PAPs and its often called active sulfate because its is the activated precursor, the yellow part is donated to some acceptor group. All of these are typically physiologically degraded in lysosomes once they are taken into appropriate cell, enzymes that break down the polymers, glycosidase, some more or less unusual disorders, mucopolysaccharidoses that one or more of these enzymes is missing. – adenine – – – 14

Extracellular Macromolecules macromolecule % carb. glycosaminoglycans (GAGs) 100 proteoglycans 90-95 glycoproteins* 2-30 fibrous proteins 1-2 * polypeptide with 1 or more oligosaccharide side chains 15

Glycoproteins: functions of glyco moieties increase protein’s solubility & hydrophilicity (sl 19) stabilize protein against denaturation proteolysis markers direct protein's destination organelle plasma membrane export (secretion) indicate protein's lifetime (sl 21) part of the protein's receptor recognition site (sl 23) signal factors such as hormones, cytokines cell-cell adhesion proteins Glycosylation: one kind of post-translational modification others: phosphorylation carboxylation Glycoproteins, third category, now substances that have lots of protein in them. The glycoproteins are very diverse in terms of function; one general property that glycation, sugars on a protein, makes them more soluble, more resistant to unfolding ,and to degradation. Glycosylation is the definition of it. Translation doesn’t put the sugars on, post translational process that does that. These glyco units serve to make the protein distinctive and a common feature, functional feature, is that they serve as part of recognition surface on a protein so that the right cell will be able to bind this protein and take it up. 16

Glycoprotein structure 17 polypeptide with 1 or more oligosaccharide side chains oligosaccharide linked to polypeptide in two ways: type linked to side chain of organelle where sugars are added to protein O-linked serine (ser), threonine (thr), Golgi complex lumen (O-glycoside) hydroxylysine (in collagen) N-linked asparagine (asn) rough ER lumen (N-glycoside) How these sugar units are linked to the protein, in yellow is polypeptide. Serine side chain, sugar unit that is linked. These are o linked, if its an oxygen. Serine threonine , or hydroxylysine, or N linked if it’s a nitrogen such as in asparagine. These are the two types of glycoproteins.

Glyco moiety structure oligosaccharide chain extends away from protein surface units mostly hexoses in pyranose (6-atom ring) form branched glycosidic links varied: α or β 1,2; 1,3; 1,4 terminal sugar often sialate 2 Typical globular protein, the folded protein is in white, then glyco part or moiety that is a side chain, different colors represent the diff monosaccharides, constituting this sugar, asapragine in blue, and t that’s the attachment part for the n linked glycoside. Also point out that the last sugar in the chain , the short chain or long ,is often sugar known sialate. It has special significance asn 7 2 7 asn 18 Stryer 4ed., p. 463

Mucins: salivary glycoproteins mol wt ~ 106 ~800 short (disaccharide) side chains terminal sugar is sialate anionic sugar at end of glyco chains of many glycoproteins very hydrophilic, extended structure ~ ~ galNAc sialate A hexose, six atoms in the ring, difference has a carboxyl group as a functional group in red. That makes sialate, means that whenever its part of a structure, ti has negative charge. Here is a protein, that has the purple circle is one sugar and the yellow triangle with minus is sialate. Here is a protein, glycoprotein that contains lots of sugars, very short side chain, only two monosacc long. This is the prototypical structure of mucins, proteins that give mucous their main property, viscous property of saliva mucous secretions in general. Consequence of these sialate and to a lesser extent the other monosaccharide, very hydrophilic, bind lots of water. Because of negative charge, very extended structure. The sialate has special properties. Other feature of these proteins, esp in saliva is that hey undergo chemical modification, usually at the hands of enzymes secreted by bacteria that have the effect of removing the sialates – 2 19

Mucins: modification & aggregation sialidase (neuraminidase) catalyzes hydrolysis of sialates from mucins secreted by oral bacteria products: less hydrophilic, less H2O-soluble, more folded, more aggregated part of the enamel pellicle & dental plaque matrix ~ ~ x H2O galNAc sialate Reaction is shown here, where hydrolysis, takes place many times, will remove the sialates and as a result, in so doing, removing lots of negative charge from this structure. In the absence of negative charges, very extended, becomes less extended then, more over because negatives are no longer there, proteins aggregate with one another, folding on one hand and aggregation on the other, these proteins in their secreted form are water soluble, don’t aggregate, as they become modified in this fashion, they precipitate, aggregate with one another, precipitate on surfaces and form the matrix of the pellicle on enamel and are important components of dental plaque that accumulates on the surface of enamel. Part of that meshwork is contributory to dental problems. This happens as a consequence of changing the mucins from their freshly secreted form, water soluble staining solution, though this less soluble form that allows them to aggregate and bind to surfaces in general. sialidase x ~ ~ 20

Role of glyco moiety in controlling protein lifetime many blood proteins have glyco chains with terminal sialate endothelial surface sialidases slowly remove sialates from these circulating proteins rate of sialate removal depends on protein's structure now-exposed gal–glcNAc… residues bind to asialoglycoprotein receptor on liver cell surface protein is then endocytosed & broken down sialoglycoprotein: sia–gal–glcNAc–[core sugars]–protein asialoglycoprotein: gal–glcNAc–[core sugars]–protein 21

both antibodies neither antibody Blood group types core sugars Type O cell surface: gal–glcNAc–gal–glc–protein† | fucose* Type A cell surface: galNAc–gal–glcNAc–gal–glc–protein† | fucose Type B cell surface: gal–gal–glcNAc–gal–glc–protein† | fucose A: have – enzyme to add galNAc to core sugars – antibody to type B antigen B: have – enzyme to add gal to core sugars – antibody to type A antigen O: have – neither enzyme AB: have – both enzymes (either galNAc or gal added to core sugars) both antibodies neither antibody 22 †or lipid * 6-deoxygalactose

Glyco moiety-binding proteins: lectins contain sites that bind specific glyco structures e.g., asialoglycoprotein receptor described on sl 21 important in intercell adhesion (i.e., lectins are CAMs: cell adhesion molecules) selectins: plasma membrane lectins that mediate cell-cell recognition & adhesion One of the slides from lecture one. By way of some of the terminology, reminder of what the sugar parts, glyco parts, glycomoiety. Some of the variety of ways in which these parts of proteins and proteins themselves function. Here is a cell surface protein, this line represents the polypeptide part, and a common way in which the carbohydrate is part of this is shown here. You never see carbohydrate parts, the glyco part inside of cells, always extracellular, non cytosolic. This means that some organelles will have glycoproteins, but sugar part will always be in lumen of ER or in lysozome. This is then a collage of the types of interactions that thee proteins can undergo. Here is a cell surface protein with carbohydrate. Virus, common infective, have proteins on their surface that are receptors for these glycoproteins. This would be the start of the process that the virus binds to the cell and infects it and causes disease. Some toxins secreted by bacteria bind to cell surface glycoproteins and that is part of the process whereby this toxin could eventually kill. The way that our own cells bind one another ins commonly by a host cel land a lymphocyte with a protein on the surface that is bound to and recognized by this type of protein. In general the proteins that bind to carbohydrate parts and lectins. Lectins: proteins that have sites analogous to active sites of enzymes. Specificity is various carbohydrate structures. An important receptor that was described two slides back, parts of cell adhesion molecules, these are part of the ways cells tick to each other and recognize each other. Pathological situations that involve these glycoproteins as well. In general, lectins, specific types are cell lectins, lectin part of that word here. In the next sections, we will have reason to see how lectins and so on work in the process of the immune system. Lehninger et al. Fig. 7-37 23