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Co-polymeric glycosaminoglycans in transformed cells. Transformation-dependent changes in the co-polymeric structure of heparan sulphate.

ABSTRACT: 1. Heparan sulphates from normal 3T3 fibroblasts are association-prone as indicated by their affinity for agarose gels substituted with cognate heparan sulphate species. Heparan sulphates from SV40-transformed or polyoma-virus-transformed cells have no affinity for the same gels. 2. Heparan sulphates from the medium, the pericellular and intracellular pools of normal, SV40-transformed and polyoma-transformed 3T3 cells were separated into four subfractions (HS1-HS4) by ion-exchange chromatography. In general, HS1-HS3 were found in cell-derived heparan sulphates, whereas HS3-HS4 were present in the medium. The heparan sulphates from transformed cells were more heterogeneous and of lower charge density than those from the normal counterpart. 3. Degradations via periodate oxidation/alkaline elimination yielded the oligomers glucosamine-(hexuronate-glucosamine)(n)-R with n=1-5 and a large proportion of N-sulphate groups. There was a large contribution of fragments n=4-5 from heparan sulphates of normal cells. These fragments were less common in low-sulphated heparan sulphates of transformed cells. In the case of medium-drived heparan sulphates all species had a low content of fragments n=4-5. 4. The size distribution of (glucuronate-N-acetylglucosamine)(n) regions was assessed after deaminative cleavage. It was broad and ranged from n=1-10 for all heparan sulphate species. In the case of medium-derived heparan sulphates there were distinct differences between normal and transformed cells. In the latter chains the N-acetyl-rich segments were both shorter and longer than in the normal case. The shape of the disaccharide peak was consistent with a lower content of O-sulphate in the heparan sulphates from transformed cells. 5. It was concluded that heparan sulphates from medium or transformed cells exhibit the greatest structural deviation from the normal case. The finding of lower proportions of extended, iduronate/glucuronate-bearing, N-sulphate-rich segments in heparan sulphates of transformed cells was particularly interesting in view of the fact that these elements have been associated with ability to self-interact.

SUBMITTER: Fransson LA

PROVIDER: S-EPMC1163631 | biostudies-other | 1982 Jan

REPOSITORIES: biostudies-other

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Project description:The structure of human skin fibroblast heparan sulphate has been examined by depolymerization with heparinase, which specifically cleaves highly sulphated disaccharides of structure GlcNSO3 (+/-6S)-alpha 1,4IdoA(2S) [N-sulphated glucosamine (6-sulphate)-alpha 1,4-iduronic acid 2-sulphate]. Heparan sulphate contained only a small proportion (approximately 10%) of linkages susceptible to this enzyme. The major products of depolymerization with heparinase were large oligosaccharides with an average molecular mass of 10 kDa (dp approximately 40, where dp is degree of polymerization; for disaccharides, dp = 2 etc.) as assessed by gel filtration on Sepharose CL-6B, compared with a molecular mass of 45 kDa (dp approximately 200) for the intact chains. The large heparinase-resistant oligosaccharides were highly susceptible to depolymerization with the enzyme heparitinase, which cleaves heparan sulphate in areas of low sulphation, where N-acetylated disaccharides [GlcNAc-alpha 1,4GlcA (N-acetylglucosaminyl-alpha 1,4-glucuronic acid)] are the predominant structural unit. Further analysis of the location of the heparinase cleavage sites indicated that they were predominantly found in a central position in GlcNSO3-alpha 1,4IdoA repeat sequences of average length four to seven disaccharides (dp 8-14). These results indicate that heparinase cleaves heparan sulphate in approximately four or five N-sulphated domains, each domain containing a cluster of two or three susceptible disaccharides; the domains are separated by long N-acetyl-rich sequences that are markedly deficient in sulphate groups. On the basis of these findings a model is proposed which depicts heparan sulphate as an ordered polymeric structure composed of an alternate arrangement of sulphate-rich and sulphate-poor regions. The sulphate-rich regions are likely to be flexible areas of the chain because of their high content of the conformationally versatile IdoA and IdoA(2S) residues. The model has important implications for the biosynthesis and functions of heparan sulphate.

Project description:1. Pig skin dermatan sulphate was degraded by periodate oxidation followed by alkaline elimination or by chondroitinase-ABC to quantify irregular repeating units, i.e. those containing D-GlcUA (D-glucuronic acid) and L-IdUA-SO4 (sulphated iduronic acid). 2. Previous results of periodate oxidation (Fransson, 1974) indicated repeating sequences in pig skin dermatan sulphate containing, on average, 3D-GlcUA, 9 L-IdUA-SO4 or 28 L-IdUA units in addition to N-acetylgalactosamine sulphate. However, complete digestion with chondroitinase-ABC yielded, at the most, 3-4 disulphated disaccharides/chain. Consequently, more than one-half of the L-IdUA-SO4 residues were present in monosulphated periods, i.e. IdUA-(SO4)-GalNAc. 3. To determine the location of L-IdUA-SO4 residues along the copolymeric chain dermatan sulphate was digested with testicular hyaluronidase. (This enzyme cleaves GalNAc-GlcUA bonds within block regions containing D-GlcUA.) By NaB3H4 reduction GalNAc residues located in the reducing end of the fragments were converted into [3H]GalNAcOH (N-acetylgalactosaminitol). Finally, the radioactive product was fragmented by periodate oxidation followed by alkaline elimination. The bulk of the radioactivity was associated with periodate-resistant oligosaccharides indicating that clusters of GlcUA-GalNAc-SO4 periods are often adjacent to a varying number of (n = 1-4) of L-IdUA-SO4-containing periods. 4. To study the distribution of L-IdUA-SO4-containing periods in relation to blocks of IdUA-GalNAc-SO4 periods different fractions of hyaluronidase-degraded dermatan sulphate were degraded separately. In all types of fragments (mol. wts. 1,500-10,000) L-IdUA-SO4-containing periods were demonstrated. In short fragments reducing terminal GalNAc-6-SO4 (6-sulphated N-acetylgalactosamine) was found confirming that these sequences were joined to relatively long D-GlcUA-containing block sequences via GalNAc-6-SO4. Moreover, low-molecular-weight oligosaccharides composed of alternating sequences were encountered. An octasaccharide derived from the carbohydrate sequence -GalNAc---GlcUA-GalNAc-IdUA-GalNAc-GlcUA-GalNAc-IdUA-GalNAc---GlcUA-GalNAc (--- indicates the position of cleavage by hyaluronidase) was identified.

Project description:Foetal human lung fibroblasts, grown in monolayer, were allowed to incorporate (35)SO(4) (2-) for various periods of time. (35)S-labelled macromolecular anionic products were isolated from the medium, a trypsin digest of the cells in monolayer and the cell residue. The various radioactive polysaccharides were identified as heparan sulphate and a galactosaminoglycan population (chondroitin sulphate and dermatan sulphate) by ion-exchange chromatography and by differential degradations with HNO(2) and chondroitinase ABC. Most of the heparan sulphate was found in the trypsin digest, whereas the galactosaminoglycan components were largely confined to the medium. Electrophoretic studies on the various (35)S-labelled galactosaminoglycans suggested the presence of a separate chondroitin sulphate component (i.e. a glucuronic acid-rich galactosaminoglycan). The (35)S-labelled galactosaminoglycans were subjected to periodate oxidation of l-iduronic acid residues followed by scission in alkali. A periodate-resistant polymer fraction was obtained, which could be degraded to disaccharides by chondroitinase AC. However, most of the (35)S-labelled galactosaminoglycans were extensively degraded by periodate oxidation-alkaline elimination. The oligosaccharides obtained were essentially resistant to chondroitinase AC, indicating that the iduronic acid-rich galactosaminoglycans (i.e. dermatan sulphate) were composed largely of repeating units containing sulphated or non-sulphated l-iduronic acid residues. The l-iduronic acid residues present in dermatan sulphate derived from the medium and the trypsin digest contained twice as much ester sulphate as did material associated with the cells. The content of d-glucuronic acid was low and similar in all three fractions. The relative distribution of glycosaminoglycans among the various fractions obtained from cultured lung fibroblasts was distinctly different from that of skin fibroblasts [Malmström, Carlstedt, Aberg & Fransson (1975) Biochem. J.151, 477-489]. Moreover, subtle differences in co-polymeric structure of dermatan sulphate isolated from the two cell types could be detected.

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Co-polymeric glycosaminoglycans in transformed cells. Transformation-dependent changes in the co-polymeric structure of heparan sulphate.

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