MOLECULAR PATHOGENESIS OF TYPE-I CONGENITAL PLASMINOGEN DEFICIENCY - EXPRESSION OF RECOMBINANT HUMAN MUTANT PLASMINOGENS IN MAMMALIAN-CELLS

We previously reported the genetic abnormality in a Japanese family wi th type I congenital plasminogen deficiency caused by a Ser(572) to Pr o(572) mutation. To characterize the molecular pathogenesis of the dis ease in this family, we expressed recombinant human wild-type and muta nt (rS572P) plasminogens in COS-1 cells. Activation-resistant wild-typ e and mutant plasminogen stable transfectants in CHO-K1. cells also we re established. Transient transfection and metabolic labeling experime nts followed by immunoprecipitation analysis showed that the mutant pl asminogen was secreted from COS-1 cells in reduced amounts, compared w ith the wild type. Endo tf digestion of the wild-type and mutant plasm inogen showed no shift in their migrations on sodium dodecyl sulfate-p olyacrylamide gel electrophoresis analysis, indicating that both conta in complex type oligosaccharide structures and could therefore be secr eted. Furthermore, the secretion of activation-resistant mutant plasmi nogen was significantly reduced. Pulse-chase experiments and Northern blot analysis showed that the impaired secretion of the mutant plasmin ogen was the consequence of the accumulation of the mutant protein ins ide the cells but not of reduced plasminogen mRNA. Immunocytochemical staining of stable transfectants also revealed that CHO-K1 cells expre ssing the activation-resistant mutant plasminogen stained mainly in th e perinuclear area, suggesting delayed processing of the mutant protei n in the intracellular transport pathway. We conclude that the impaire d secretion of mutant plasminogen, due to intracellular accumulation, is the molecular pathogenesis of type I congenital plasminogen deficie ncy caused by a Ser(572) to Pro(572) mutation. (C) 1997 by The America n Society of Hematology.