Endoproteolytic processing of the dibasic cleavage site in the human protein C precursor in transfected mammalian cells: effects of sequence alterations on efficiency of cleavage.
The human protein C precursor undergoes extensive co- and posttranslational modification during its biosynthesis in the liver. These modifications include glycosylation, gamma-carboxylation and beta-hydroxylation of specific amino acids, and endoproteolytic processing to remove the pre- and propeptides and also to remove the pair of basic amino acids that connect the light and heavy chains in the precursor. Specific molecular signals have been elucidated which direct several of these modifications; however, the mechanism for cleavage and removal of the basic amino acid pair has not been established. In the present study, a recombinant mammalian expression system has been used to study the molecular signals that direct removal of this basic amino acid pair. Mutations were introduced by site-directed mutagenesis either to insert additional basic amino acids or to alter the sequence adjacent to the basic pair by point mutations. The mutant protein precursors were expressed and analyzed for the degree of processing to 2-chain form and also for the location of the cleavage site (by N-terminal sequencing) and subsequent removal of the basic amino acids from the newly formed C terminus of the light chain. These experiments have shown that human protein C can be readily synthesized and secreted in several mammalian cell lines. However, cell lines vary considerably in their capacity to remove the dibasic pair in the protein C precursor and, like the liver, secrete a mixed population of 1-chain and 2-chain forms of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Foster
D C
DC
Zymogenetics, Inc., Seattle, Washington 98105.
Sprecher
C A
CA
Holly
R D
RD
Gambee
J E
JE
Walker
K M
KM
Kumar
A A
AA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003488:Cyanogen Bromide; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007163:Immunosorbent Techniques; D008969:Molecular Sequence Data; D009154:Mutation; D011486:Protein C / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity; D014162:Transfection
D011486:Protein C; D011498:Protein Precursors; D010450:Endopeptidases; D003488:Cyanogen Bromide