Peter Hovingh

The heparitin sulfates (heparan sulfates).

Heparitin sulfate has been isolated from several sources, namely: a commercial lung polysaccharide preparation, beef lung, beef aorta, human amyloid liver, human intestine, and urine of a patient with mucopolysaccharidosis. The polysaccharides isolated were extensively purified, fractionated, and characterized. The data obtained show that heparitin sulfate is not a single compound but constitutes a family of related polymers which differ in sulfate content and in the arrangement of charged groups. These compounds are readily distinguished from most other glycosamino-glycuronans (mucopolysaccharides) by composition and optical rotation. They can be differentiated from heparin by their content of sulfate and N-acetyl groups, and D-glucuronic acid residues and by the ratio of the carbazole to orcinol uronic acid values. Due to the variations of charge distribution and molecular size, the heparitin sulfates are found to a considerable extent in admixture with other acidic polysaccharides during isolation and fractionation procedures. The particular type of heparitin sulfate obtained varies considerably with the tissue of origin. The lung-derived material was found to contain the largest range of subfractions. The heparitins isolated from aorta and amyloid liver were fairly homogenous in themselves, but differed from each other.

The disaccharide repeating-units of heparan sulfate

Abstract Five disaccharides have been isolated after degradation of heparan sulfate by heparinase (heparin lyase) and heparitinase (heparan sulfate lyase) and are suggested to represent the repeating units of the polysaccharide. They all contain a 4,5-unsaturated uronic acid residue and are: (a) A trisulfated disaccharide that is apparently identical to a disaccharide repeating-unit of heparin; (b) a disulfated disaccharide that seems unique for heparan sulfate and contains 2-deoxy-2-sulfamidoglucose and uronic acid sulfate residues; (c) a nonsulfated disaccharide containing a 2-acetamido-2-deoxyglucose residue; (d) a monosulfated disaccharide containing a 2-acetamido-2-deoxyglucose sulfate residue; and (e) a monosulfated disaccharide containing a 2-deoxy-2-sulfamidoglucose residue. Yields of these disaccharides from different heparan sulfate fractions are discussed in relation to possible arrangements of these units in the intact polymer.

Structural studies of heparitin sulfates.

Heparitin sulfate fractions with a large range in sulfate content were subjected to degradation by Flavobacterium heparinase and by nitrous acid. The products obtained were fractionated by chromatography, characterized, and used to arrive at tentative structures for these complex polysaccharides. The heparitin sulfate chains examined appear to be composed of: 1. uninterrupted blocks of N-acetylglucosamine containing disaccharides; 2. larger blocks with a molecular weight range of 5000 to 6000 which include the N-acetyl block but do not contain heparinase sensitive linkages; 3. segments containing mainly areas where N-acetyl, N-sulfate and some disulfated units alternate in the chain. The size and arrangement of these polymer segments seem to vary with the sulfate content of a particular heparitin sulfate. For instance, the polysaccharides with the highest degree of sulfation do not appear to contain N-acetyl blocks of significant size.

Structural studies on heparin. Tetrasaccharides obtained by heparinase degradation

Three tetrasaccharides representing major structural sequences of heparin were isolated in good yield and characterized after degradation of heparin by purified flavobacterial heparinase. N-Desulfation was necessary to achieve good separation of these closely related compounds from each other. One of the tetrasaccharides was shown to be derived from the fully sulfated repeating segments; to contain L-iduronic acid and six sulfate groups, and have the structure delta 4,5- HexpA -(2-SO4)-(1----4)-alpha-D- GlcpN -(N-SO4)-(6-SO4)-(1- ---4)-alpha -L- IdopA -(2-SO4)-(1----4)-D- GlcN -(N-SO4)-(6-SO4). The second contained a D-glucuronic acid unit that was nonsulfated instead of the L-iduronic acid, and the third, obtained in a fairly low yield, contained five sulfate groups, three of which being located on the disaccharide at the nonreducing end, and having the structure delta 4,5- HexpA -(2-SO4)-(1----4)-alpha-D- GlcpN -(N-SO4)-(6-SO4)-(1- ---4)-alpha -L- IdopA -(2-SO4)-(1----4)-D- GlcN -(N-SO4). All tetrasaccharides had a sulfated, unsaturated uronic acid unit at the nonreducing end, confirming that the heparinase requires sulfated L-iduronic acid units for activity.

The enzymatic degradation of heparitin sulfate. II. Isolation and characterization of non-sulfated oligosaccharides.

Abstract A non-sulfated, N-acetylated disaccharide was isolated from hepatirin sulfate after enzymatic digestion. This compound containing α, β unsaturated uronic acid is an isomer of a disaccharide obtained previously from hyaluronic acid. It contains an α- d -(1 → 4) rather than the β- d -(1 → 3) linkage found in the hyaluronate compound. The isolation of another non-sulfated, N-acetylated compound, most likely a tetrasaccharide, indicates that at least two or more N-acetylated units occur in sequence in the polysaccharide. The linkages in the non-sulfated portion of heparitin sulfate appear to be mainly α- d -(1 → 4) .

Glycosaminoglycans in Anodonta californiensis, a Freshwater Mussel

The synthesis of glycosaminoglycans (GAG) in a freshwater mussel was studied in organ culture using labeled precursors. The major GAGs synthesized were determined and characterized by chemical and enzymatic methods. They were shown to be heparin and an unusual type of heparan sulfate. Gills produced about 50% of each polymer; mantles synthesized little heparin and mostly the heparan-sulfate-like compound, which is similar to a GAG isolated previously from lobsters. No significant amounts of chondroitin sulfates were present. Histological data showed that the sulfate-labeled GAGs were present mainly in exterior pericellular and basement membrane locations of gills and mantle. That is, they would be in contact with the external aqueous environment, suggesting a potential role in calcium transport and storage.

Glycosaminoglycans in two mollusks, Aplysia californica and Helix aspersa, and in the leech, Nephelopsis obscura

Abstract The presence of glycosaminoglycans was examined in two mollusks (Pulmonates): the terrestrial garden snail, Helix aspersa, and the opishtobranchian sea slug, Aplysia californica and also in the leech (Hirudinea, Erpobdellidae, Nephelopsis obscura ). Organs in the garden snail contained predominately chondroitin sulfate and heparan sulfate as a lesser component. The ctenidium of the sea slug contained mainly chondroitin sulfate and a compound which migrated on electrophoresis as heparin but additional data indicated that it could also represent a highly sulfated form of heparan sulfate. The foregut contained only the heparin-like polymer. No standard glycosaminoglycan could be identified in the leech although a polydispersed polysaccharide containing uronic acid, hexosamine and sulfate was shown to be present. A detailed analysis of the heparan sulfate isolated from the garden snail is also given.

Proteoglycan and glycosaminoglycan free chain expression in keratinocytes, endothelium, and mesenchymal cells

Cultured fibroblasts, bovine aortic endothelial cells, and human keratinocytes synthesize both proteoglycans and glycosaminoglycan free chains, the proportions varying between cell types. The major metabolic labeling is in proteoglycans, except for keratinocytes with approximately 60% of product as free chains. The proteoglycans range from approximately 50- greater than 1000 kDa, and the glycosaminoglycan side chains derived by alkaline elimination are approximately 30- greater than 100 kDa. The glycosaminoglycan free chains, in contrast, are smaller, from approximately 7-40 kDa in mass. The proteoglycans are both medium and cell layer constituents, whereas the glycosaminoglycan free chains are essentially confined to cells. The cellular proteoglycans and a portion of the free chains are accessible to in situ digestion by Flavobacterial glycosaminoglycan lyases, presumably reflecting localization to the cell surface. Collectively, the data show the free chains to be a common feature of all cells studied and to be partly expressed on cell surfaces. We hypothesize that the processing that creates these free chains occurs on cell surfaces, in which location they could serve ligand receptor functions.

DIVERGENT REGULATION OF PROTEOGLYCAN AND GLYCOSAMINOGLYCAN FREE CHAIN EXPRESSION IN HUMAN KERATINOCYTES AND MELANOCYTES

SummaryKeratinocytes and melanocytes, which together form units of structure and function within human epidermis, are known to differ in expression of autocrine growth factors, particularly those with heparin binding affinity. Because such cytokines could be regulated by the endogenous heparinlike glycosaminoglycan, heparan sulfate, proteoglycan synthesis was compared between human keratinocytes and melanocytes cultured from a common donor. Following steady-state isotopic labeling under conditions of active growth (low density cultures) and growth inhibition (high density cultures), the sulfated polymers were isolated from conditioned media and cell extracts. We found that keratinocytes produced substantially more sulfated glycosaminoglycans than did the melanocytes. There was no evidence for hyaluronic acid synthesis by the melanocytes. The majority of [35S]-sulfate labeling was in the heparan sulfates of the keratinocytes and in the chondroitin sulfates of the melanocytes. During the transition from active growth to growth inhibition, there was increased heparan sulfate proteoglycan and free chain synthesis by keratinocytes but not by melanocytes, and chondroitin sulfate proteoglycan production declined in both cell lineages. The differences may reflect divergent evolution as each cell type came to exploit those complex polysaccharides in different ways to regulate molecular pathways of growth and differentiation. The coupling of growth inhibition with augmented synthesis of heparan sulfates observed for the keratinocytes suggests a regulatory role in growth factor signaling in that cell type.

The digestion of cell surface-associated glycosaminoglycans by crude and purified flavobacter heparinum enzymes: Disparate effects on cell adhesion

The effects of crude and purified glycosaminoglycan (GAG) lyases on cell adhesion to plastic substrates were studied in three established cell lines. In the presence of crude heparinase, BALB/c 3T3 and B16.F10 melanoma cells and a fibrosarcoma line were markedly inhibited in their ability to attach and spread on two types of tissue culture-grade plastic. The crude enzyme effect was dose dependent, reversible, and co-eluted with heparinase activity by Sepharose Cl-6B chromatography. Boiling of the preparation, however, did not eliminate its effect. Furthermore, none of the purified GAG lyases which were tested reproduced the effect of the crude preparation. Therefore, our results indicate that although GAG lyases are effective in digesting the GAGs of cell surfaces, the removal of these substances has no perceptible effect on the substrate adhesion of the three cell types evaluated.