THE NATURE OF A COVALENT LINKAGE BETWEEN CHITIN AND PROTEIN IN THE HORSESHOE CRAB, LIMULUS POLYPHEMUS (AMINO ACIDS, SUGARS, PEPTIDES)

A covalently bonded chitin-protein (residual) complex from Limulus polyphemus was isolated by treating shell material with alkali. This chitinous "residue" contained 1.8% protein. In agreement with earlier studies its amino acid composition was found to be high in glycine, tyrosine, proline, and valine, and no methionine or cysteine was found. A high performance liquid chromatographic method identified mannose, galactose, and rhamnose as the principal neutral sugar components of this residual complex. Limited amounts of glucose and fucose were also detected.;The compositions of these chitin-derived peptides (and glycopeptides), including amino acids, neutral sugars, and amino sugars, were determined. Four of the five peptides were shown to be glycopeptides containing two moles of glucosamine each. None of the peptides contained neutral sugar. The molecular weights of the peptides, estimated on the basis of amino acid and glucosamine content, ranged from 1,800 to 4,000 daltons.;The smallest of the chitin-derived peptides (pep-1), contained only 12 amino acids, and was digested with carboxypeptidase to partially sequence the peptide. The amino acid sequence at the carboxy-terminal was determined to be: -alanine-glutamine-tyrosine-glycine-valine-lysine-COOH;Chemical methods were used to distinguish among the different proposed chitin-protein linkages. These tests showed that the glucosamine-peptide linkage is N-glycosidic, and hence that the chitin-protein linkage in N-glycosidic. Based on the composition and partial amino acid sequence of pep-1, the linkage point was identified as asparagine.;Enzymatic methods were used to produce water-soluble glycoproteins by cleavage of the repeating polysaccharide of this Limulus chitin fraction. The chitin glycoproteins were cleaved with trypsin, and the resulting peptides and glycopeptides were isolated and purified using high performance liquid chromatography.;On the basis of amino acid composition, Limulus chitin was identified as being significantly different from other chitins. This difference may arise from the role protein plays in cross-linking the Limulus chitin.