Isolation, biochemical and immunological characterisation of two sea urchin glycoproteins bearing sulphated poly(sialic acid) polysaccharides rich in N-glycolyl neuraminic acid.
Two different sialoproteins were isolated from the sea urchin shell by guanidine hydrochloride extraction in the presence of Triton X-100. The sialoproteins (SP I and SP II) were purified on DEAE-Sephacel and Sepharose CL-6B and separated from each other by density gradient centrifugation. The ratio between recovered SP I and SP II was 1:4.5 and their M(r)s 650 and 600 kDa, respectively. They were degraded by neuraminidase, endoglycosidase F and peptide N-glycosidase F resulting in fragments of similar relative molecular mass (M(r)s). Although their protein cores have approximately the same relative molecular mass of 500 kDa, they differ markedly in their contents of aspartic acid/asparagine, glycine, leucine and phenylalanine, as well as in the primary amino acid sequence of their N-terminal peptides. Carbohydrate analyses showed that the sialic acid content was higher in SP I (11.4% of dry tissue weight) than in the more prominent SP II (5.3%). Two types of carbohydrates, O-glycosidically-linked polysaccharides and N-glycosidically-linked oligosaccharides are present in both sialoproteins. SP I contains 10-11 polysaccharide chains whereas SP II contains 5-6. The polysaccharides are linked to protein cores via galactosamine, have approximately the same M(r) of 12 kDa and contain 32-33 N-glycolyl neuraminic acid, 10-11 glucosamine, 6-7 sulphate and 6-8 neutral monosaccharide residues. Sialic acid residues are organized in a poly(sialic acid) unit which is present in the non-reducing terminal of the polysaccharides and degraded by neuraminidase. Hexosamines, sulphates and neutral monosaccharides are all constituents of the sialic acid free region of the chain near the reducing end. Two oligosaccharide populations were isolated from SP I, one major (70% of the total oligosaccharides) with M(r) of approximately 3 kDa and the other with M(r) of 1.5 kDa. In SP II, however, only a 3-kDa oligosaccharide population was present. The oligosaccharides from both sialoproteins are N-glycosidically linked to asparagine via the glucosamine and contain mannose, glucosamine, galactosamine and sialic acids. Antibodies against SP II were raised in rabbits and it was shown that the antigenicity of SP II was lost on either neuraminidase or trypsin digestion, indicating that both the poly(sialic acid) units of the polysaccharide and the protein core are antigenically active. As expected, SP II showed considerable cross-reactivity with SP I due to the common poly(sialic acid) structure. There were no significant reactivities of SP II and SP I with antibodies to bovine bone sialoprotein and osteopontin. The biological role of the two sea urchin sialoproteins as developmentally regulated products of the tissue remains to be elucidated.
Karamanos
N K
NK
Department of Chemistry, University of Patras, Greece.
Manouras
A
A
Anagnostides
S
S
Makatsori
E
E
Tsegenidis
T
T
Antonopoulos
C A
CA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000606:Amino Sugars / Q000032:analysis; D000818:Animals; D002417:Cattle; D004722:Endoribonucleases; D000939:Epitopes / Q000032:analysis; D006026:Glycoside Hydrolases; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009005:Monosaccharides / Q000032:analysis; D019158:N-Acetylneuraminic Acid / Q000032:analysis / Q000737:chemistry; D009438:Neuraminic Acids / Q000032:analysis*; D009439:Neuraminidase; D009844:Oligosaccharides / Q000032:analysis; D012260:Ribonucleases; D012617:Sea Urchins / Q000737:chemistry*; D017421:Sequence Analysis; D012795:Sialoglycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D013045:Species Specificity; D013431:Sulfates / Q000032:analysis
D000596:Amino Acids; D000606:Amino Sugars; D000939:Epitopes; D009005:Monosaccharides; D009438:Neuraminic Acids; D009844:Oligosaccharides; D012795:Sialoglycoproteins; D013431:Sulfates; C032592:N-glycolylneuraminic acid; D004722:Endoribonucleases; D012260:Ribonucleases; C034663:ppp(A2'p)nA-dependent endoribonuclease protein, mouse; D006026:Glycoside Hydrolases; C090534:endo-alpha-sialidase; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid