Purification and characterization of a natural agglutinin from the serum of the hermit crab Diogenes affinis.
A natural agglutinin from the serum of the hermit crab Diogenes affinis was purified to homogeneity by a single-step affinity chromatography using N-acetylglucosamine-coupled Sepharose 6B. The purified serum agglutinin (PSA) showed a strong affinity for rat RBC, and its hemagglutinating (HA) activity was specifically dependent on Ca2+ and reversibly sensitive to EDTA. PSA in active form has a molecular mass estimate of 185 kDa and is composed of four non-identical subunits (51, 49, 42 and 39 kDa) cross-linked by interchain disulfide bonds. The homogeneity of PSA was corroborated by immunodiffusion and immunoelectrophoretic analyses using rabbit antiserum raised against the agglutinin. The antibodies in this antiserum appear to be specific for RBC-binding sites of the agglutinin molecules as revealed by the ability of the antiserum to neutralize HA activities of both whole serum and PSA of D. affinis. In HA-inhibition assays performed with several carbohydrates and glycoproteins, PSA showed a distinct and unique specificity for acetyl group in carbohydrates independently of the presence of this group on C-2 or C-5 and its stereochemical arrangement in the axial or equatorial orientation. Besides, this agglutinin appears to recognize the terminal N- and O- acetyl groups in the oligosaccharide chain of glycoconjugates. The HA activity of D. affinis agglutinin was also susceptible to inhibition by lipopolysaccharides from diverse gram-negative bacteria, which might indicate a significant in vivo role of this humoral agglutinin in the host immune response against bacterial infections.
Murali
S
S
Department of Zoology, University of Madras, Guindy Campus, India.
Mullainadhan
P
P
Arumugam
M
M
D000373:Agglutinins / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*; D000818:Animals; D033381:Anomura / Q000378:metabolism*; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006386:Hemagglutination Tests; D006801:Humans; D007106:Immune Sera; D005779:Immunodiffusion; D051381:Rats
D000373:Agglutinins; D007106:Immune Sera