Heparan Sulfate Sulfation Research Articles

High-performance liquid chromatographic identification of eight constitutional disaccharides from heparan sulfate isomers digested with heparitinases.

Identification with specific heparan sulfate-lyases, heparitinase I and heparinase of the constitutional unsaturated disaccharide (delta Di-SHS) derived from heparan sulfate (HS) isomers and heparin was achieved using high-performance liquid chromatography (HPLC) with a sulfonated styrene-divinylbenzene copolymer. Eight delta Di-SHS products derived from HS isomers were identified. Enzymatic digestion with heparitinase I and heparinase converts heterogeneous sulfated HS isomers and heparin into different delta Di-SHS. The practical application of these enzymes was examined using specific enzymes and HPLC. In a patient with Hurler syndrome, eight individual delta i-SHS were identified in urinary HS isomers.

Characterization of a cell mutant specifically defective in the synthesis of anticoagulantly active heparan sulfate.

We have investigated the characteristics of clonal L cell mutant VI-7 that has previously been demonstrated to exhibit reduced levels of cell surface proteoglycans containing heparan sulfate (HS) with regions of defined monosaccharide sequence that interact with antithrombin (HSact) (de Agostini, A. L., Lau, H. K., Leone, C., Youssoufian, H., and Rosenberg, R. D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9784-9788). Pulse labeling revealed that the synthesis of HSact in mutant cells, as compared to wild-type cells, was reduced by 5-7-fold, which is identical to the previously observed reduction in cell surface-bound antithrombin. This alteration is independent of growth state since production of HSact by mutant cells, as compared to wild-type cells, is decreased to a similar extent in exponentially growing and post-confluent cultures. The synthetic defect was specific for HSact since production of total HS and chondroitin sulfate by mutant cells, as compared to wild-type cells, was identical in magnitude. The synthetic abnormality is not due to an alteration in core protein since complementation was not observed when mutant cells were stably transfected with the epitope-tagged ryudocan cDNA which can initiate HSact production. Structural analyses revealed that HS from mutant cells, as compared to wild-type cells, exhibited normal molecular weight, extent and distribution of sulfate, and disaccharide composition, which indicate that the mutation did not affect HS biosynthetic enzymes. Together, the data suggest that mutant VI-7 is defective in a regulatory component which directly or indirectly coordinates HS biosynthetic enzymes to specifically generate the defined monosaccharide sequence of HSact.

Liver heparan sulfate structure. A novel molecular design

The structure of rat liver heparan sulfate (HS) has been investigated using a combination of (a) chain scission with specific reagents, (b) disaccharide compositional analysis, and (c) end-referenced sequence analysis of the proximal, protein-linked region of the chain. This study reveals that the liver synthesizes a highly sulfated HS species (1.34 sulfates/disaccharide), particularly high in N-sulfation (60%) and 2-O-sulfate content (36%). Approximately half of the latter is found in trisulfated disaccharides, i.e. IdceA(2-OSO3) alpha 1-4GlcNSO3 (6-OSO3). End-referencing methodology established the existence of an extended, unmodified heparan (GlcUA beta 1-4GlcNAc) sequence, 8-11 disaccharides in length, attached to the linkage tetrasaccharide, similar to that found in a number of other HS species. Directly following this is a mixed HexUA1-4GlcNR(6-OSO3) (where GlcNR represents alpha-D-glucosamine with an unspecified N-substituent)-containing sequence of variable length, culminating in the appearance of the first IdceA(2-OSO3) residue approximately 20 disaccharides from the linkage region, i.e. approximately 40% along the length of the chain. The distal 60% of the polysaccharide is highly sulfated (approximately 2 sulfates/disaccharide) and mainly comprises three heparin-like domains, highly enriched in IdceA(2-OSO3) residues. Overall, liver HS qualifies as an extreme member of the HS family, with a considerable proportion of heparin-like structure asymmetrically concentrated to the distal part of the chain.

Concurrent reduction in the sulfation of heparan sulfate and basement membrane assembly in a cell model system

Basement membranes (BMs) are specialized extracellular matrices that have important roles in cell attachment, migration, growth and differentiation. The murine teratocarcinoma cell line, M1536-B3, has been shown to produce a model BM composed of laminin, entactin and heparan sulfate proteoglycans but lacking collagen. Therefore, M1536-B3 cells are an excellent model system in which to study the role of non-collagenous components in BM assembly. We have used these cells to test for a requirement of mature heparan sulfate (HS) chains in BM assembly. Growth of M1536-B3 cells in the presence of chlorate, an inhibitor of activated sulfate synthesis, resulted in a dose-dependent decrease in the sulfation of glycosaminoglycans and reduction in the charge density of the isolated HS. The undersulfated HS from chlorate-treated cells had a decreased binding capacity for laminin when compared with control HS. Concurrent with these changes in sulfation, chlorate treatment of M1536-B3 cells resulted in the failure of BM assembly, which was restored upon removal of the chlorate from the growth medium. These results were not due to major alterations in cell attachment, spreading, growth, protein synthesis, or to an inability of the cells to synthesize and secrete laminin. These data suggest that the sulfation of HS and its subsequent ability to interact with other BM components play major roles in the assembly and structure of BMs.

Changes in Glycosaminoglycans During the Neuritogenesis in PC12 Pheochromocytoma Cells Induced by Nerve Growth Factor

Previously, we had suggested that heparan sulfate (HS) makes some contribution to a flat-shaped morphology of PC12D cells. Therefore, we carried out quantitative and qualitative analyses of glycosaminoglycans (GAGs), the polysaccharide moiety of proteoglycans, during neuritogenesis in PC12 cells that is induced by nerve growth factor (NGF). (a) In PC12 cells, NGF induced a flat-shaped morphology with a few short processes after 3 days of culture, and then it elicited short and long neurites after 6 (in approximately 30% of cells) and 9 (in 60-70%) days of culture, respectively. (b) HS and chondroitin sulfate (CS) were detected in the cell layer at all times. Only CS was found in the medium at 3 and 6 days, whereas a low level of HS, in addition to CS, was detectable on day 9. (c) In the NGF-treated cultures, the amounts of cell-associated HS per cell were two to three times as high as those in the respective nontreated cultures at all times, whereas the amount based on phospholipid was about twofold higher after 3 days of culture. (d) The levels of HS labeled with [35S]sulfate during the last 48 h of the culture were 1.5- to twofold higher in the NGF-treated cultures than in the respective controls at any time. (e) The amount of cell-associated CS per cell (or per unit of phospholipid), but not of labeled CS per cell, was transiently enhanced at 3 days in culture with or without NGF.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuronal proteoglycans: biosynthesis and functional interaction with neurons in vitro

Glycosaminoglycans (GAGs) with electrophoretic mobilities on cellulose acetate similar to heparin (H), heparan sulfate (HS), and chondroitin sulfate (CS) were detected in cell extracts and in conditioned medium of high-density, neuron-enriched cultures labeled with 35SO4. Heparitinase digestion revealed that heparan sulfate proteoglycans (HSPGs) were heterogeneous in charge density and were responsible for neurite outgrowth activity for sensory neurons in conditioned medium. In the presence of beta-D-xyloside, an inhibitor of proteoglycan assembly, there was an increase of released GAGs with the mobility of CS and heavily sulfated HS but a decrease in neurite outgrowth activity on a laminin substrate at times greater than 14 hr. In the presence of beta-D-xyloside or the monoclonal antibody HNK-1 (Leu 7), which recognizes a neuronal cell surface epitope, there was a time-dependent inhibition of process formation that was half-maximal at 7-8 hr and independent of laminin concentration or cell adhesion to the laminin substrate. The kinetics and magnitude of the inhibitory effects of beta-D-xyloside and HNK-1 (Leu 7) were similar, and the influence of HNK-1 (Leu 7) could no longer be observed in the presence of beta-D-xyloside. Pretreatment of the laminin substrate with conditioned medium from high-density neuron cultures resulted in an increased rate of neurite formation compared with untreated laminin. Where the laminin substrate had been pretreated with conditioned medium, maximal inhibition by HNK-1 (Leu 7) was apparent from the earliest times. However, if the conditioned medium had been digested with heparitinase, neither enhanced neurite outgrowth nor the inhibitory influence of HNK-1 (Leu 7) were observed. The present biosynthetic and functional studies suggest that neurons are one source of HSPGs. These data complement and extend earlier studies suggesting a role for HSPGs in neurite formation. The experiments also provide evidence for modulation of laminin by HSPGs which interact with laminin and promote neurite outgrowth that is mediated by a cell surface receptor at or in juxtaposition to the HNK-1 (Leu 7) epitope.

Isolation and characterization of peptidoglycans in urine from patients with mucopolysaccharidoses

1. 1. Urinary dermatan sulfate (DS) and heparan sulfate (HS) were purified from mucopolysaccharidosis patients. 2. 2. DS shows average mol. wt 8600–12,000 (approx. one half of tissue DS), iduronic acid content 99.1–99.6% (81.8% in tissue DS), core peptide mostly di- or tri-peptide (—Ser—Gly— or —Ser—Gly—Glu—). 3. 3. Molecular weight of HS ranged from 2500 to 20,000, averaging about 5000. 4. 4. Highly sulfated HS was found in the low molecular weight fraction, and no bound core peptide. 5. 5. By contrast, HS in the high molecular weight fraction bound one sulfate per repeating unit, which include core peptide.