Isolation of plaice (Pleuronectes platessa) α1-microglobulin:: conservation of structure and chromophore

A cDNA coding for plaice (Pleuronectes platessa) alpha(1)-microglobulin (Leaver et al., 1994, Comp. Biochem. Physiol. 108B, 275-281) was expressed and purified from baculovirus-infected insect cells. Specific monoclonal antibodies were then prepared and used to isolate the protein from plaice liver and serum. Mature 28.5 kDa alpha(1)-microglobulin was found in both liver and serum. The protein consisted of an 184 amino acid peptide with a complex N-glycan in position Asn(123), one intrachain disulfide bridge and a yellow-brown chromophore. Physicochemical characterization indicated a globular shape with a frictional ratio of 1.37, electrophoretic charge-heterogeneity and antiparallel beta-sheet structure. A smaller, incompletely glycosylated, yellow-brown alpha(1)-microglobulin as well as a 45 kDa precursor protein were also found in liver. The chromophore was found to be linked to alpha(1)-microglobulin intracellularly. Recombinant plaice alpha(1)-microglobulin isolated from insect cells had the same N-terminal sequence, globular shape and yellow-brown color as mature alpha(1)-microglobulin, but carried a smaller, fucosylated, non-sialylated N-glycan in the Asn(123) position. The concentration of alpha(1)-microglobulin in plaice serum was 20 mg/l and it was found both as a 28.5 kDa component and as high molecular weight components. Thus, the size, shape, charge and color of plaice alpha(1)-microglobulin were similar to mammalian alpha(1)-microglobulin, indicating a high degree of structural conservation between fish and human alpha(1)-microglobulin. The monoclonal antibodies against plaice alpha(1)-microglobulin cross-reacted with human alpha(1)-microglobulin, emphasizing the structural similarity. (C) 1999 Elsevier Science B.V. All rights reserved.