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

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Abstract

A cDNA coding for plaice (Pleuronectes platessa) α1-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 α1-microglobulin was found in both liver and serum. The protein consisted of an 184 amino acid peptide with a complex N-glycan in position Asn123, 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 β-sheet structure. A smaller, incompletely glycosylated, yellow-brown α1-microglobulin as well as a 45 kDa precursor protein were also found in liver. The chromophore was found to be linked to α1-microglobulin intracellularly. Recombinant plaice α1-microglobulin isolated from insect cells had the same N-terminal sequence, globular shape and yellow-brown color as mature α1-microglobulin, but carried a smaller, fucosylated, non-sialylated N-glycan in the Asn123 position. The concentration of α1-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 α1-microglobulin were similar to mammalian α1-microglobulin, indicating a high degree of structural conservation between fish and human α1-microglobulin. The monoclonal antibodies against plaice α1-microglobulin cross-reacted with human α1-microglobulin, emphasizing the structural similarity. Copyright (C) 1999 Elsevier Science B.V.

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Subject classification (UKÄ) – MANDATORY

  • Cell and Molecular Biology

Keywords

  • α-Microglobulin, Baculovirus, cDNA, Glycosylation, Liver, Serum, Protein HC, Teleost
Original languageEnglish
Pages (from-to)222-233
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Volume1430
Issue number2
Publication statusPublished - 1999 Mar 19
Publication categoryResearch
Peer-reviewedYes