Expression and characterization of apolipoprotein(a) kringle IV types 1, 2 and 10 in mammalian cells

• Published in: Protein engineering Vol. 7; no. 5; p. 723
• Main Authors: Sangrar, W, Marcovina, S M, Koschinsky, M L
• Format: Journal Article
• Language: English
• Published: England 01-05-1994
• Subjects: Amino Acid Sequence; Animals; Apolipoproteins A - chemistry; Apolipoproteins A - genetics; Apolipoproteins A - metabolism; Base Sequence; Blotting, Northern; Cell Line; Embryo, Mammalian; Enzyme-Linked Immunosorbent Assay; Fibrinogen - metabolism; Glycosylation; Humans; Immunosorbent Techniques; Kringles; Lysine - metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Sepharose - metabolism; Structure-Activity Relationship; Transfection
• ISSN: 0269-2139

We have designed expression constructs containing sequences corresponding to apolipoprotein(a) kringle IV types 1, 2 and 10 and used these constructs to transfect human embryonic kidney cells. We have also expressed a mutant form of kringle IV type 2 in which the N-linked glycosylation site has been removed by replacement of an asparagine residue with an alanine. Immunoprecipitation analysis of [35S]Cys-labeled transfected cell culture supernatants resulted in the observation of two bands for kringle IV type 1 (M(r) approximately 30,000 and 26,000), two bands for kringle IV type 2 (M(r) approximately 25,000 and 22,000), two bands for kringle IV type 10 (M(r) approximately 27,000 and 23,000) and one band for the glycosylation mutant (M(r) approximately 22,000). In all cases, observed molecular weights greatly exceeded those predicted from amino acid sequence, suggesting the presence of both N- and O-linked glycans. None of the recombinant single kringles were observed to bind to fibrinogen as determined by ELISA or by co-immunoprecipitation in the case of kringle IV type 10 and only kringle IV type 10 was able to bind to lysine--Sepharose. These data suggest that apo(a) binding to fibrinogen/fibrin may require motif(s) in addition to apo(a) kringle IV type 10.