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

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Abstract
We have designed expression constructs containing sequences corresponding to apolipoprotein(a) kringle FV types 1, 2 and 10 and used these constructs to transfect human embryonic kidney cells. We have also expressed a mutant form of kringle FV 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 (Mr ∼30 000 and 26 000), two bands for kringle IV type 2 (Mr ∼25 000 and 22 000), two bands for kringle IV type 10 (Mr ∼27 000 and 23 000) and one band for the glycosylation mutant (Mr ∼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.

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