Chondroitin sulfate cluster of epiphycan from salmon nasal cartilage defines binding specificity to collagens.

Epiphycan (EPY) from salmon nasal cartilage has a glycosaminoglycan (GAG) domain that is heavily modified by chondroitin 4-sulfate and chondroitin 6-sulfate. The functional role of the GAG domain has not been investigated. The interaction of EPY with collagen was examined in vitro using surface plasmon resonance analysis. EPY was found to bind to type I collagen via clustered chondroitin sulfate (CS), while a single chain of CS was unable to bind. Types I, III, VII, VIII and X collagen showed high binding affinity with EPY, whereas types II, IV, V, VI and IX showed low binding affinities. Chemical modification of lysine residues in collagen decreased the affinity with the clustered CS. These results suggest that lysine residues of collagen are involved in the interaction with the clustered CS, and the difference in lysine modification defines the binding affinity to EPY. The clustered CS was also involved in an inter-saccharide interaction, and formed self-associated EPY. CS of EPY promoted fibril formation of type I collagen.

Epiphycan from salmon nasal cartilage is a novel type of large leucine-rich proteoglycan.

Chum salmon (Oncorhynchus keta) nasal cartilage was examined by next-generation DNA sequencing and mass spectrometric analyses, and 14 types of proteoglycans including epiphycan (EPY) were found. A cDNA encoding EPY was cloned and sequenced. The cDNA encoded 589 amino acids comprised a glycosaminoglycan (GAG) domain containing 55 potential GAG-modified sites (Ser-Gly and/or Gly-Ser), a cysteine cluster and 6 leucine-rich repeats. EPY was purified from salmon nasal cartilage and the structure of the GAG was characterized. As a result of unsaturated disaccharide analysis, GAG was found to be composed of chondroitin 6-sulfate (58.0%), chondroitin 4-sulfate (26.5%) and non-sulfated chondroitin (15.3%). The average molecular weight of GAG was estimated to be 3.0 × 10(4). Ser-100 and Ser-103 were identified as serine residues substituted by GAG chains by chemical modification and mass spectrometric analysis. More than 50 serine residues were assumed to be substituted by GAG chains. EPY is heavily substituted by chondroitin sulfate, giving an overall molecular weight of just under 2 × 10(6). EPY from salmon nasal cartilage is a novel type of large leucine-rich proteoglycan.