Bruce Caterson

Keratan sulfate proteoglycan during embryonic development of the chicken cornea

Antibodies to corneal keratan sulfate proteoglycan (KSPG) were used to characterize the pattern of KSPG accumulation during differentiation of neural crest cells in the stroma of embryonic chick cornea. Immunohistochemistry with monoclonal antibody I22 to keratan sulfate found this KSPG antigen localized inside stromal cells at stage 29 (Day 6), ca. 12 hr after migration into the primary stroma. A 2- to 3-day lag then occurred before appearance of extracellular keratan sulfate, first seen on Day 9 (Stage 35) in the posterior stroma. Keratan sulfate antigen accumulated in a posterior to anterior direction during subsequent development. Uniform staining of the stroma for keratan sulfate did not occur until after Day 16. Among several tissues, only corneal stroma contained an extracellular matrix which stained for keratan sulfate, though intracellular staining of some cartilage cells was observed. Accumulation of KSPG antigens in developing cornea was measured in unfractionated guanidine extracts with a quantitative ELISA using three different antibodies against KSPG. Increases were first detected after Day 9 using monoclonal I22, and somewhat later with the other two antibodies. Assays with all three antibodies detected a sustained, exponential increase of KSPG throughout the 5 days prior to hatching. Keratan sulfate continued to accumulate after hatching, but an antibody with specificity to KSPG core protein, detected no relative increase in antigen after hatching. This suggests a modulation of KSPG primary structure late in development and after hatching. Overt differentiation of individual neural crest cells thus appears to begin ca. 12 hr after their arrival in the primary stroma; a lag of 2-3 days precedes active secretion of KSPG.

Demonstration of a keratan sulfate proteoglycan and a mannose-rich glycoconjugate in corpora amylacea of the brain by immunocytochemical and lectin-binding methods.

The aim of the present report is to call attention to the fact that corpora amylacea (CA) of the brain have similar biochemical and immunological characteristics as the connective tissue matrix. We have demonstrated that CA reacted positively toward a monoclonal antibody that specifically recognized the polysaccharide determinant on cartilage proteoglycans. Material strongly bound to concanavalin A was demonstrated in CA, blood vessel wall and cartilage and suggested high levels of mannose in these structures. These findings are in agreement with the known fact that both brain and cartilage contain keratan sulfate and high mannose glycoproteins. We propose that CA may result from accumulation of glycoconjugates normally present in the brain tissue matrix as the result of aging.

Immunochemical characterization and ultrastructural localization of chondroitin sulfates and keratan sulfate in embryonic chick bone marrow

SummaryMonoclonal antibodies directed against specific carbohydrate epitopes on chondroitin 4-/dermatan sulfate, chondroitin 6-sulfate, keratan sulfate, and a monoclonal antibody directed against the hyaluronate binding region were used to characterize proteoglycans extracted from embryonic chick bone marrow. About half of the proteoglycans separate into the high density fraction on a CsCl gradient. Glycosaminoglycan-specific antibodies recognize proteoglycans from all fractions; this includes an antibody directed against keratan sulfate. Some proteoglycans, principally in the high buoyant density fraction, contain sites recognized by the antibody specific for the hyaluronate binding region. Within limits of detection, all core proteins belong to the high-molecular-weight category, with weights in excess of 212 kD. Antibodies directed against chondroitin 4-/dermatan sulfate and against keratan sulfate primarily bind to extracellular matrix material located in the extracellular spaces and to matrix elements in the pericellular regions of fibroblastic stromal cells. The antibody that recognizes chondroitin 6-sulfate binds to sites on surfaces of fibroblastic stromal cells and also to extracellular matrix material. Little or no antibody binding is detected on surfaces of granulocytic cells. These studies indicate that chondroitin sulfate and keratan sulfate chains are both present in the proteoglycan extract.

The structures of N- and O-glycosidic carbohydrate chains of a chondroitin sulfate proteoglycan isolated from the media of the human aorta

A large Mr chondroitin sulfate proteoglycan was extracted from the media of human aorta under dissociative conditions and purified by density-gradient centrifugation, ion-exchange chromatography, and gel filtration chromatography. Removal of a contaminating dermatan sulfate proteoglycan was accomplished by reduction, alkylation and rechromatography on the gel filtration column. After chondroitinase ABC treatment, the proteoglycan core was separated from a residual heparan sulfate proteoglycan by a third gel filtration chromatography step. As assessed by radioimmunoassay, the isolated proteoglycan core was free of link protein, but possessed epitopes that were recognized by antisera against the hyaluronic acid binding region of bovine cartilage proteoglycan as well as those that were weakly recognized by anti-keratan sulfate antisera. Following beta-elimination of the protein core, the liberated low Mr oligosaccharides were partially resolved by Sephadex G-50 chromatography, and their primary structure was determined by 500-MHz1H NMR spectroscopy in combination with compositional sugar analysis. The N-glycosidic carbohydrate chains, which were obtained as glycopeptides, were all biantennary glycans containing NeuAc and Fuc; microheterogeneity in the NeuAc----Gal linkage was detected in one of the branches. The N-glycosidic glycans have the following overall structure: (Formula: see text). The majority of the O-glycosidic carbohydrate chains bound to the protein core were found to be of the mucin type. They were obtained as glycopeptides and oligosaccharide alditols, and possessed the following structures: NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol, [NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol, and NeuAc alpha-(2----3) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol. The remainder of the O-glycosidic carbohydrate chains bound to the isolated proteoglycan were the hexasaccharide link regions of the chondroitin sulfate chains that remained after chondroitinase ABC treatment of the native molecule. These latter glycans, which were obtained as oligosaccharide alditols, had the following structure (with GalNAc free of sulfate or containing sulfate bound at either C-4 or C-6): delta 4,5GlcUA beta(1----3)GalNAc beta(1----4)GlcUA beta(1----3)Gal beta(1----3)Gal beta(1----4)Xyl-ol.

Chondroitin, chondroitin 6-sulphate, chondroitin 4-sulphate and dermatan sulphate proteoglycans in normal and pathological human muscle ☆

Chondroitin, chondroitin 6-sulphate, chondroitin 4-sulphate and dermatan sulphate proteoglycans were immunolocalized by monoclonal antibodies applied to human muscle sections digested with chondroitinase. In normal muscle the 4 proteoglycans presented a different extracellular localization: unsulphated chondroitin sulphate (chondroitin) was present in the endomysium and around capillaries, chondroitin 6-sulphate in the basal membrane zone, chondroitin 4-sulphate in the vessel adventitia, in the endomysium around capillaries and, to a lesser degree, in the perimysium, dermatan sulphate in the perimysium and, to a lesser extent, in the vessel adventitia and in the endomysium around capillaries. The enlarged endomysium of pathological muscle contained chondroitin and chondroitin 4-sulphate. Chondroitin 6-sulphate and dermatan sulphate did not seem present in the increased connective tissue. No peculiar pattern was observed in the various neuromuscular diseases studied. The specific extracellular distribution, the different biochemical composition and the different ability to bind to other extracellular components suggest a different biological role of these compounds. Chondroitin 6-sulphate is a component of a highly specialized extracellular structure, namely basal membrane. Chondroitin and chondroitin 4-sulphate participate in the composition of actively changing extracellular matrix such as the endomysium in pathological muscle. On the other hand, dermatan sulphate is a constituent of the perimysium that is a more static extracellular structure.

The phosphorylation of human link proteins

Three link proteins of 48,44 and 40 kDa were purified from human articular cartilage and identified with monoclonal anti-link protein antibody 8-A-4. Two sets of lower molecular weight proteins of 30-31 kDa and 24-26 kDa also contained link protein epitopes recognized by the monoclonal antibody and were most likely degradative products of the intact link proteins. The link proteins of 48 and 40 kDa were identified as phosphoproteins while the 44 kDa link protein did not contain 32P. The phosphorylated 48 and 40 kDa link proteins contained approximately 2 moles PO4/mole link protein.