Effect of low-density lipoproteins on the synthesis and secretion of proteoglycans by human endothelial cells in culture.

We studied the effect of low-density lipoproteins (LDL) on the synthesis and secretion of proteoglycans by cultured human umbilical-vein endothelial cells. Confluent cultures were incubated with [35S]sulphate or [3H]glucosamine in lipoprotein-deficient serum in the presence and in the absence (control) of LDL (100-400 micrograms/ml), and metabolically labelled proteoglycans in culture medium and cell layer were analysed. LDL increased accumulation of labelled proteoglycans in medium and cell fractions up to a concentration of 200 micrograms/ml. At this concentration of LDL the accumulations of proteoglycans in medium and cell layer were 65% and 32% respectively above control for 35S-labelled proteoglycans, and 55% and 28% respectively above control for 3H-labelled proteoglycans. At concentrations above this LDL was found to depress the accumulation of proteoglycans in medium and cell layer. Gel filtration on Sepharose CL-4B showed that in both control and LDL-treated cultures the cell layer contained a large (Kav. = 0) and a small (Kav. = 0.35) heparan sulphate proteoglycan, whereas the culture medium contained a large heparan sulphate proteoglycan (Kav. = 0) and a smaller isomeric chondroitin sulphate proteoglycan (control, Kav. = 0.35; LDL-treated, Kav. = 0.17). The relative increase in hydrodynamic size of the isomeric chondroitin sulphate proteoglycan (Mr 150,000 compared with 90,000) in the medium of cultures exposed to LDL was partly attributable to the larger size of the glycosaminoglycan side chains (Mr 39,000 compared with 21,000). The isomeric chondroitin sulphate proteoglycan in LDL-treated culture was relatively enriched in chondroitin 6-sulphate compared with that in control cultures (39% compared with 29%). Pulse-chase studies showed that LDL treatment did not alter the turnover rate of proteoglycans as compared with controls, implying that the elevation in proteoglycan accumulation in LDL-treated cultures was due to enhanced synthesis. These results demonstrate that LDL can modulate proteoglycan synthesis by cultured vascular endothelial cells, resulting in the secretion of a larger isomeric chondroitin sulphate proteoglycan enriched in chondroitin 6-sulphate.     

Purification and characterization of human platelet proteoglycan.

Freshly prepared platelets were shown to contain glycosaminoglycans equivalent to 530 micrograms of hexuronate/10(11) platelets. When the platelets were extracted with 4 M-guanidinium chloride containing proteinase inhibitors, and the extract was dialysed extensively against 7 M-urea solution, almost all of proteoglycan was recovered in the urea-soluble fraction. The proteoglycan was purified from the urea-soluble fraction with a yield of 47% by DEAE-Sephacel chromatography, CsCl-density-gradient centrifugation, Bio-Gel A-15m gel filtration and then rechromatography on DEAE-Sephacel. The purified proteoglycan contained 30% glucuronic acid, 32% N-acetylgalactosamine, 14% sulphate and 15% protein. Serine, glutamic acid, glycine, aspartic acid and leucine accounted for 64% of the total amino acids. The Mr of the proteoglycan was assessed to be approx. 136000 by sedimentation-equilibrium methods. The galactosaminoglycan released by alkaline-borohydride treatment of the proteoglycan was converted stoichiometrically into 4-sulphated unsaturated disaccharide by digestion with chondroitinase AC-II, indicating that the galactosaminoglycan was fully sulphated chondroitin 4-sulphate. The apparent Mr of the chondroitin sulphate was assessed to be 28000 by gel filtration on Bio-Gel A-0.5m (KD 0.18). On two-dimensional electrophoresis on a cellulose acetate membrane, the chondroitin sulphate gave a single compact spot co-migrating with a reference chondroitin sulphate, indicating that the chondroitin sulphate chains were homogeneous in both length and charge density. On the basis of these results, the proteoglycan in human platelets was concluded to be a macromolecule of Mr 136000 containing four chondroitin 4-sulphate chains each with the apparent Mr of 28000.     

Gossypol inhibition of acrosin and proacrosin, and oocyte penetration by human spermatozoa

Gossypol, a known antispermatogenic agent, was found to effectively inhibit the highly purified boar sperm proacrosin-acrosin proteinase enzyme system by irreversibly preventing the autoproteolytic conversion of proacrosin to acrosin and reversibly inhibiting acrosin activity. The agent appears to prevent the self-catalyzed by not the acrosin-catalyzed activation of proacrosin. In additional experiments, brief exposure of human semen to concentrations of gossypol, which did not visibly alter spermatozoal motility or forward progression, was found to irreversibly inhibit the conversion of proacrosin to acrosin although the activity of the nonzymogen acrosin was not decreased, and also to prevent the human spermatozoa from penetrating denuded hamster oocytes. Gossypol inhibition of proacrosin conversion to acrosin closely paralleled the decline in oocyte penetration. Racemic (+/-) gossypol was equally as effective as the enantiomer (+) gossypol. The results suggest that the inhibition of proacrosin conversion to acrosin is a mechanism by which gossypol exerts its antifertility effect at nonspermicidal concentrations and that low levels of gossypol should be tested for their contraceptive action when placed vaginally.     

Activation of proacrosin by a locally produced component of human follicular fluid

Components of human follicular fluid were separated on Sepharose 6B columns and the effects of different fractions on the conversion of pig proacrosin to acrosin were examined. A high-molecular-weight fraction (Mr greater than 3,000,000) of follicular fluid was a potent stimulator of this reaction. The proacrosin converting activity was absent in the corresponding fraction of blood serum. The acceleration of proacrosin activation was dependent on the concentration of material with proacrosin converting activity. The results indicate that human follicular fluid contains a high-molecular-weight component of local origin which is capable of accelerating proacrosin in a dose-dependent manner.     

Proacrosin/acrosin during guinea pig spermatogenesis

Enriched populations of guinea pig spermatogenic cells were isolated by sedimentation velocity at unit gravity. Each cell population was analyzed for the presence of members of the proacrosin/acrosin family by enzymography, immunoblotting, and immunofluorescence. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis in gels containing 0.1% gelatin, protease activities with molecular weights of 55,000 (major) and 50,000 (minor) were detected in round spermatid extracts. Condensing spermatid extracts contained protease activities with molecular weights between 55,000 and 50,000. These major protease activities had molecular weights similar to antigens detected by immunoblotting with a monospecific rabbit antiserum directed against purified boar acrosin. Extracts of guinea pig sperm and the soluble acrosomal components released following the acrosome reaction induced with ionophore A23187 contained three major protease activities (Mr 32,000, 34,000, 47,000) but only the 47,000 Mr protease cross-reacted with the antibody. The spermatid and sperm protease activities were inhibited and activated by classical effectors of acrosin activity from other species. Immunofluorescence demonstrated that proacrosin/acrosin was present as early as the Golgi phase of spermiogenesis. In addition, immunoreactivity was confined to the acrosomes in a manner characteristic of each spermatid stage. These results demonstrate that proacrosin/acrosin can be detected in the earliest spermiogenic stages by electrophoretic and immunological techniques and suggest that changes in the molecular weights of proacrosin/acrosin occur as spermatids mature.     

Studies on the rabbit proacrosin-acrosin system

A single molecular form (Mr = 68,000 approx) of a homogeneous preparation of rabbit testis proacrosin (S. K. Mukerji and S. Meizel (1979) J. Biol. Chem. 254, 117;21-11728) was initially converted by autoactivation into an acrosin (Mr = 68,000); both gave a single activity and protein bands with similar electrophoretic mobilities (Rm = 0.25) when subjected to polyacrylamide disc gel electrophoresis on 7.5% gel at pH 4.5. Two additional bands (Rm values of 0.395-0.412 and 0.497-0.519, respectively) were noticeable only when proacrosin was activated further after attaining maximum activity. The slowest- and the fastest-moving bands were separated into two acrosin activity peaks by Sephadex G-100 gel-filtration chromatography on a calibrated column. The molecular weights of the two proteins, determined by rechromatography on the same column, was estimated to be 68,000 and 34,000, respectively. Also, sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis of three acrosins gave protein bands which corresponded to molecular weights of approximately 68,000, 52,000, and 34,000, respectively. Electrophoresis data suggest that the loss of acrosin activity generally observed following prolonged activation of proacrosin is caused by self-aggregation of the Mr 34,000 form of acrosin. This property was not shown by Mr 68,000 acrosin. Initial acrosin (Mr = 68,000) was activated by divalent cations such as Ca2+ and Mg2+. The enzyme was inhibited by Zn2+, Fe2+, Hg2+, and sulfhydryl blockers such as 5,5'-dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate, and iodoacetate, apparently due to their reaction with one out of six titratable sulfhydryl groups per mole of acrosin. Probably Zn2+ is involved in acrosomal stabilization. The initial rabbit acrosin (Mr = 68,000) appears to be the major and most stable form, and is generated from proacrosin with little structural alteration. This may be the functionally active form which plays an essential role in mammalian fertilization.     

Aortic endothelial cells synthesize a large chondroitin sulphate proteoglycan capable of binding to hyaluronate.

Confluent cultures of mouse aortic endothelial (END-D) were incubated with either [35S]methionine or 35SO4 2-, and the radiolabelled proteoglycans in media and cell layers were analysed for their hyaluronate-binding activity. The proteoglycan subfraction which bound to hyaluronate accounted for about 18% (media) and 10% (cell layers) of the total 35S radioactivity of each proteoglycan fraction. The bound proteoglycan molecules could be dissociated from the aggregates either by digestion with hyaluronate lyase or by treatment with hyaluronate decasaccharides. Digestion of [methionine-35S]proteoglycans with chondroitinase and/or heparitinase, followed by SDS/polyacrylamide-gel electrophoresis, indicated that the medium and cell layer contain at least three chondroitin sulphate proteoglycans, one dermatan sulphate proteoglycan, and two heparan sulphate proteoglycans which differ from one another in the size of core molecules. Among these, only the hydrodynamically large chondroitin sulphate species with an Mr 550,000 core molecule was shown to bind to hyaluronate. A very similar chondroitin sulphate proteoglycan capable of binding to hyaluronate was also found in cultures of calf pulmonary arterial endothelial cells (A.T.C.C. CCL 209). These observations, together with the known effects of hyaluronate on various cellular activities, suggest the existence of possible specialized functions of this proteoglycan subspecies in cellular processes characteristic of vascular development and diseases.     

Guinea pig proacrosin is synthesized principally by round spermatids and contains O-linked as well as N-linked oligosaccharide side chains

Proacrosin is the zymogen precursor of acrosin, a sperm protease believed to play an essential role in fertilization. In this study, we used primary cultures of guinea pig spermatogenic cells to examine the temporal appearance and mechanisms of synthesis and processing of proacrosin during acrosome development. Following [35S]methionine incorporation and immunoprecipitation, cultured spermatogenic cells were found to synthesize two forms of proacrosin (Mr 54,000 and 57,000). Proacrosin was synthesized mainly by round spermatids. By immunoblotting, proacrosin became very prominent in round spermatids and persisted throughout spermiogenesis. Pulse-chase experiments demonstrated that the Mr 54,000 form of proacrosin was converted to the Mr 57,000 form, presumably reflecting posttranslational processing of carbohydrate side chains. When spermatogenic cells were cultured in the presence of tunicamycin, the synthesized proacrosin had an Mr of 54,000. However, in vitro translation of mRNA extracted from guinea pig testis followed by immunoprecipitation indicated that the core polypeptide of proacrosin has an Mr of 44,000. Guinea pig spermatogenic cells incorporated glucosamine and fucose into the oligosaccharides of proacrosin. Treatment of guinea pig testis proacrosin with N-glycosidase or O-glycosidase reduced the Mr by 3-7%. These results indicate that proacrosin is synthesized by postmeiotic cells and the enzyme contains N- and O-linked oligosaccharides.     

Benzamidine as an inhibitor of proacrosin activation in bull sperm.

Epididymal and ejaculated sperm contain a zymogen form of acrosin (acrosomal proteinase, EC 3.4.21.10) which is converted to active enzyme prior to fertilization. Benzamidine at concentrations greater than 10 mM has been shown to inhibit the conversion of proacrosin to acrosin. Based on this inhibition, a procedure was developed for extracting and quantitating the proacrosin content of bull sperm. Sperm were isolated from semen and washed by centrifugation through 1.3 M sucrose and the outer acrosomal membrane removed by homogenization. When 25 mM benzamidine was added to the semen and wash solutions, 98% or more of the acrosin activity in the sperm homogenate was present as proacrosin. Proacrosin can be extracted from the sperm homogenate by dialysis at pH 3, which solubilized the proenzyme and removed benzamidine. Benzamidine has been useful in isolating proacrosin and provides a new method for studying the activation of proacrosin in intact sperm. Neutralization of sperm extracts, after removal of benzamidine, resulted in rapid activation of proacrosin with a pH optimum of 8.5, and activation was complete within 15 min over a pH range of 7.0 to 9.5. Rapid activation also occurred during the washing of sperm in the absence of benzamidine, and this activation correlated with a swelling of the acrosomal membrane. This rapid activation appears to result from a small amount of acrosin activity consistently present in the sperm extract. These results indicate an autocatalytic conversion of proacrosin to acrosin and suggest that disruption of the acrosomal membrane may trigger this activation.     

Comparative activation studies with extracted and purified human proacrosin

Proacrosin was purified from acid extracts of human spermatozoa by concanavalin A precipitation and Bio-Gel P-100 chromatography. Two molecular weight forms of proacrosin were obtained, a major one with a Mr of 70,000-71,000 and a minor one with a Mr of 47,000-53,000. In contrast to sperm extracts, the purified forms of proacrosin were free of acrosin inhibitor(s) and nonzymogen acrosin. By modulating pH, ionic strength and temperature, the activation of proacrosin in sperm extracts was compared to only the major form of purified proacrosin, since it seemed to be the source of the lower molecular weight form of proacrosin. In both preparations, proacrosin activation occurred maximally over a broad pH range (7.6-8.8 for purified proacrosin and 7.6-9.6 for extract). Additionally, an ionic strength of 0.1 and above caused a decrease in proacrosin activation in both preparations. Similarly, proacrosin was sensitive to short incubation periods at 45 degrees C and above which caused a decrease in the amount of proacrosin found in both preparations.     

Separation and characterization of two populations of aggregating proteoglycans from cartilage.

Intermediary gel immunoelectrophoresis was used to show that purified aggregating cartilage proteoglycans from 2-year-old steers contain two distinct populations of molecules and that only one of these is immunologically related to non-aggregating cartilage proteoglycans. The two types of aggregating proteoglycans were purified by density-gradient centrifugation in 3.5M-CsCl/4M-guanidinium chloride and separated by zonal rate centrifugation in sucrose gradients. The higher-buoyant-density faster-sedimenting proteoglycan represented 43% of the proteoglycans in the extract. It had a weight-average Mr of 3.5 X 10(6), did not contain a well-defined keratan sulphate-rich region, had a quantitatively dominant chondroitin sulphate-rich region and contained 5.9% protein and 23% hexosamine. The lower-buoyant-density, more slowly sedimenting, proteoglycan represented 15% of the proteoglycans in the extract. It had a weight-average Mr of 1.3 X 10(6), contained both the keratan sulphate-rich and the chondroitin sulphate-rich regions and contained 7.3% protein and 23% hexosamine. Each of the proteoglycan preparations showed only one band on agarose/polyacrylamide-gel electrophoresis. The larger proteoglycan had a lower mobility than the smaller. The distribution of chondroitin sulphate chains along the chondroitin sulphate-rich region was similar for the two types of proteoglycans. The somewhat larger chondroitin sulphate chains of the larger proteoglycan could not alone account for the larger size of the proteoglycan. Peptide patterns after trypsin digestion of the proteoglycans showed great similarities, although the presence of a few peptides not shared by both populations indicates that the core proteins are partially different.     

Cell-free translation of messenger RNA for chondroitin sulphate proteoglycan core protein in rat cartilage.

Total RNA was extracted from the cartilage tissues rat Swarm chondrosarcoma, neonatal-rat breastplate and embryonic-chicken sterna and translated in wheat-germ cell-free reactions. The core protein of the chondroitin sulphate proteoglycan subunit was identified among translation products of rat mRNA by its apparent Mr of 330 000 and by its immunoprecipitation with specific antisera prepared against rat or chicken proteoglycan antigens. The apparent Mr of the rat proteoglycan core protein is 8000-10000 less than that of the equivalent chicken cartilage core-protein product.     

Isolation, characterization and properties of the oversulphated chondroitin sulphate proteoglycan from squid skin with peculiar glycosaminoglycan sulphation pattern.

Oversulphated chondroitin sulphate proteoglycan from squid skin was isolated from 4 M guanidine hydrochloride extract by ion-exchange chromatography, gel chromatography and density gradient centrifugation. The proteoglycan had Mr 3.5 x 10(5), contained on average six oversulphated chondroitin sulphate chains (Mr 4 x 10(4)) bound on a polypeptide of Mr 2.8 x 10(4), and oligosaccharides consisting of both hexosamines, glucuronic acid, sulphates and fucose as the only neutral monosaccharide. The major amino acids of the proteoglycan protein core are glycine (corresponding to about one third of the total amino acids), aspartic acid/asparagine and serine, together amounting to 50% of the total. The proteoglycan was resistant to the proteolytic enzymes V8 protease, trypsin (treated with diphenylcarbamoyl chloride), alpha-chymotrypsin and pronase, while it was completely degraded by papain and to a large extent by collagenase. Pretreated proteoglycan with chondroitinase AC was degraded by pronase to a large extent and slightly by V8 protease and trypsin. The proteoglycan did not interact with hyaluronic acid and did not form self-aggregates. Oversulphated chondroitin sulphate chains were composed of unusual sulphated disaccharide units which were isolated and characterized by HPLC. In particular, it contained 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose 4-sulphate (delta di-4S) and disulphated disaccharides (delta di-diS) [90% 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid 2/3-sulphate)-D-galactose 6-sulphate (delta di-diSD) and 10% 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid 2/3-sulphate)-D-galactose 4-sulphate (delta di-diSK)] as the major disaccharides, significant amounts of trisulphated disaccharides (delta di-triS) and small amounts of 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose 6-sulphate (delta di-6S) and 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose (delta di-OS). Trisulphated disaccharides contained sulphate groups at C-4 and C-6 of the galactosamine and at C-2 or C-3 of the glucuronic acid. By HPLC analysis of a pure preparation of oversulphated chondroitin sulphate, it was found that it contains glucose, galactose, mannose and fucose most likely as branches.     

Proacrosin conversion inhibitor. Purification and initial characterization of a boar sperm protein which prevents the conversion of proacrosin into acrosin

A proacrosin conversion inhibitor present in boar spermatozoa has been purified and initially characterized. Purification methods included sequential acid extractions of washed spermatozoa at pH 4.0, pH 3.5, and pH 2.5 followed by successive gel filtrations of the pH 2.5 sperm extract supernatant over Sephadex G-75 and G-50. The resulting 8.8-fold purified materials were judged to be homogeneous by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis, had an estimated molecular weight of 12,800, and a constant specific activity of 65 units/mg. Treatment with the proteinases acrosin, trypsin, or chymotrypsin destroyed the highly purified proacrosin conversion inhibitor, indicating that it is a protein. Additional properties of the inhibitor included stability to long periods of storage at pH 3.0 and 4 degrees C, stability to boiling and lyophilization, and an absolute requirement for divalent cations to maintain activity. The highly purified proacrosin conversion inhibitor does not inhibit acrosin. Therefore, it apparently acts to prevent proacrosin conversion by selectively inhibiting the zymogen's self-catalyzed conversion mechanism.     

Effects of various conditions of semen storage on the acrosin system of human spermatozoa

Stability of the human sperm acrosin system (major components: non-zymogen acrosin, proacrosin and acrosin inhibitor) was studied under various conditions of semen storage used clinically or in the laboratory. Freezing at -196 degrees C caused a profound decrease in total acrosin content and in the amount of this enzyme present in zymogen form (proacrosin), but resulted in some increase in non-zymogen acrosin. Acrosin inhibitor did not appear to be significantly affected by this treatment. No relationship was present between the decreases in sperm motility induced by freezing to -196 degrees C and the alterations in total acrosin, proacrosin and non-zymogen acrosin. Storage of whole semen at -20 degrees C had deleterious effects on all the components of the acrosin system measured except for non-zymogen acrosin. Major decreases in the total acrosin, proacrosin and acrosin inhibitor occurred after only 1 day at -20 degrees C and continued slowly thereafter. Whole semen kept at room temperature for up to 24 h after ejaculation did not show any significant changes in the sperm acrosin system. Seminal plasma did not have a detrimental or stabilizing effect of acrosin and proacrosin when spermatozoa were kept at room temperature. However, removal of seminal plasma and re-suspension of spermatozoa in 0.9% NaCl resulted n the liberation of a significant amount of the acrosin inhibitor from the spermatozoa and the apparent activation of some of the proacrosin to acrosin.     

A Glycoprotein Expressed by Human Fibrous Astrocytes is a Hyaluronate-Binding Protein and a Member of the CD44 Family

We have isolated and characterized an antigen from normal human brain called p80, so called because it migrated with an Mr of 80 kDa on SDS PAGE. The Mr of 80 kDa consists of a protein of about 55-60 kDa and carbohydrate (20-25 kDa). The carbohydrate is almost entirely of the N-linked type, although a small amount of O-linked carbohydrate was detected. Cross-reactivity with monoclonal antibodies A3D8 and A1G3 showed that p80 could therefore be considered an isoform of the CD44 adhesion molecules. In addition, specific binding to hyaluronate which was not competed for by proteoglycan demonstrated that it involved different sites than the proteoglycan binding sites. We also observed that fucoidan and dextran sulphate increased the binding by 200-250% while chondroitin sulphate C also increased the binding but to a lesser extent. Heparin, heparan sulphate and chondroitin sulphates A and B did not have such an effect. The binding of p80 to hyaluronate was pH dependent with a maximum at pH 6.4. We concluded that p80 was an astrocyte specific adhesion molecule.     

Characterisation of chondroitin/dermatan sulphate proteoglycans synthesised by rat mammary myoepithelial and fibroblastic cell lines.

Chondroitin sulphate proteoglycans were isolated from the culture medium of rat mammary gland fibroblast (Rama 27) and myoepithelial (Rama 401) cell lines which had been labelled with [35S]sulphate. Chromatography on Sepharose CL-4B indicated that the Rama 401 proteoglycan was larger than the Rama 27 proteoglycan (Kav values 0.47 and 0.56, respectively). Treatment of the proteoglycans with alkaline NaBH4 yielded chondroitin sulphate chains with average M(r) values of 37,000 (Rama 401) and 21,000 (Rama 27). Structural analysis of the glycosaminoglycan chains indicated that both were co-polymers of chondroitin and dermatan sulphate although there were differences in the amounts and distribution of the disaccharide repeating units. The M(r) values of the core proteins, determined by immunoblotting, were about 43,000 and 46,000 (Rama 27) and 44,500 (Rama 401). Using an antibody to chondroitin sulphate proteoglycan in immunofluorescence experiments, the proteoglycan was demonstrated on the surface of both cell lines. Rama 27 cells additionally possessed an extensive fibrous extracellular matrix which also stained with the antibody. Staining of sections of lactating mammary gland suggested that the proteoglycan was present in the basement membrane as well as the stromal connective tissue. The presence of chondroitin sulphate proteoglycan in the basement membrane was confirmed by ultrastructural immunolocalisation.     

Structural studies on sialylated and sulphated O-glycosidic mannose-linked oligosaccharides in the chondroitin sulphate proteoglycan of brain.

We have previously described the structures of neutral and sialylated O-glycosidic mannose-linked tetrasaccharides and keratan sulphate polysaccharide chains in the chondroitin sulphate proteoglycan of brain. The present paper provides information on a series of related sialylated and/or sulphated tri- to penta-saccharides released by alkaline-borohydride treatment of the proteoglycan glycopeptides. The oligosaccharides were fractionated by ion-exchange chromatography and gel filtration, and their structural properties were studied by methylation analysis and fast-atom-bombardment mass spectrometry. Five fractions containing [35S]sulphate-labelled oligosaccharides were obtained by ion-exchange chromatography, each of which was eluted from Sephadex G-50 as two well-separated peaks. The apparent Mr values of both the large- and small-molecular-size fractions increased with increasing acidity (and sulphate labelling) of the oligosaccharides. The larger-molecular-size fractions contained short mannose-linked keratan sulphate chains of Mr 3000-4500, together with some asparagine-linked oligosaccharides. The smaller tri- to penta-saccharides, of Mr 800-1400, appear to have a common GlcNac(beta 1-3)Manol core, and to contain one to two residues of sialic acid and/or sulphate.     

Studies on the polydispersity and heterogeneity of cartilage proteoglycans. Identification of 3 proteoglycan structures in bovine nasal cartilage.

1. Three chondroitin sulphate components were isolated from adult bovine nasal cartilage after treatment with alkaline NaB3H. Average molecular weights of 13000, 18 600 and 28 000 were obtained for chondroitin sulphate species representing 10, 52 and 38% (w/w) of the total chondroitin sulphate respectively. Each chondroitin sulphate pool has a narrow molecular-weight distribution. 2. A proteoglycan subunit preparation, isolated from one nasal cartilage by extraction and density-gradient fractionation in dissociative solvents, partitioned on a CSCl density gradient according to size and composition. Variation of proteoglycan molecular weight across the gradient was directly related to the average chondrotin sulphate chain length, which in turn reflected the relative proportion of the three chondroitin sulphate pools in each proteoglycan fraction. Consideration of proteoglycan molecular parameters, compositions and behaviour on sedimentation leads to a proposal that nasal cartilage contains 3 distinct proteoglycan pools, each of which has a constant number of chondroitin sulphate side chains of different average molecular weight. 3. Molecular-weight distribution parameters for these proteoglycan preparations indicate that all serine residues on the protein core capable of initiating chondroitin sulphate biosynthesis are occupied and that proteoglycan polydispersity results directly from the polydispersity of the attached chondroitin sulphate component.     

Activation and subsequent degradation of proacrosin is mediated by zona pellucida glycoproteins, negatively charged polysaccharides, and DNA

Boar proacrosin (E.C. 3.4.21.10, Mw 53 kD) was isolated by a modified method and subjected to autoactivation. Previously described molecular intermediates of 49 and 43 kD and a stable form (beta-acrosin, 35 kD) were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoactivation was expedited in the presence of either zona pellucida glycoproteins, fucoidan, or DNA. The end point of this accelerated conversion was the complete degradation of otherwise stable beta-acrosin via the formation of a characteristic active intermediate protein of 30 kD. All intermediate molecular forms observed during proacrosin activation/conversion exhibited the N-terminal sequence of the boar acrosin heavy chain, indicating a C-terminal processing mechanism. Hence zona pellucida glycoproteins stimulate proacrosin activation as well as acrosin degradation. Such a mechanism of proenzyme activation and degradation is to our knowledge described here for the first time and points to a previously unrecognized role of zona pellucida during gamete interaction.