ABSTRACT
A new type of hyaluronidase was isolated from squid cranial cartilage. The enzyme seems to be localised extracellularly, since it is extracted from the tissue by 0.5 M sodium acetate, pH 7.0, in the presence of proteinase inhibitors. Degradation studies suggest that the enzyme belongs to the family of endoglycosidases generating oligosaccharides of rather large size. The best activity of the enzyme was observed at pH 7.0 and 37 degrees C and the optimum buffer for digestion was 0.15 M Tris acetate. It is inactive in sodium phosphate, morpholine acetate and HEPES buffers. The enzyme degrades aggrecan, hyaluronan, chondroitin sulphate and oversulphated chondroitin sulphate.
Subject(s)
Cartilage/enzymology , Decapodiformes/enzymology , Hyaluronoglucosaminidase/metabolism , Skull/enzymology , Animals , Chondroitin Sulfates/metabolism , Extracellular Fluid/enzymology , Hyaluronoglucosaminidase/analysis , Hyaluronoglucosaminidase/isolation & purificationABSTRACT
The three populations of squid cranial cartilage proteoglycans, D1D1A, D1D1B and D1D2 appeared to have a high degree of polydispersity. Gel electrophoresis and immunoblotting analysis showed that polydispersity was mainly due to the variable size of chondroitin sulphate E chains. This was further ascertained after rotary shadowing electron microscopy of proteoglycan core proteins and glycosaminoglycan side chains and statistical analysis of the sizes measured for both components. Enzymic treatment of the proteoglycan core proteins produced different peptides from each population, suggesting that the observed heterogeneity of the proteoglycans is due to their core proteins. Antibodies were raised in rabbits against all proteoglycans and enzyme-linked immunosorbent analysis of proteoglycan core proteins revealed that the proteoglycans, even heterogeneous, shared many common epitopes. Part of the common proteoglycan epitopes were found to be located in chondroitin sulphate E chains. Heterogeneity of squid proteoglycans was also investigated by studying their interactions with collagen and it was found that only the two populations of high molecular mass, D1D1A and D1D2, were able to interact with only collagen type I, the latter stronger than the former.
Subject(s)
Cartilage/chemistry , Proteoglycans/chemistry , Proteoglycans/ultrastructure , Animals , Blotting, Western , Chondroitin ABC Lyase , Decapodiformes , Electrophoresis, Agar Gel , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Microscopy, ElectronABSTRACT
Squid cranial cartilage extracts were found to contain a protein with a molecular mass of 35 kDa immunoreacting with an antiserum against sheep link protein. Because hyaluronan is not detected in this tissue and the structure of proteoglycans is different to that of aggrecan or versican, this observation was studied further. The 35 kDa protein was purified from cartilage extracts and immunolocalised in Western blots by both the polyclonal antibody and the mAb 8A4. It was found that it was able to bind to hyaluronan and to aggrecan. Direct and competitive microplate binding experiments showed that the squid protein binds to G1 domain of aggrecan, similarly to cartilage link protein and, therefore, it could be a link-like protein molecule of squid cranial cartilage. The 35 kDa protein was also able to bind to squid proteoglycan and this suggested that it might participate in squid cartilage proteoglycan aggregate formation.
