In previous studies have shown that the interaction between factor IXa and VIII involves the light chain of factor VIII and that this interaction inhibited by the monoclonal antibody CLB-CAg A against the factor VIII region Gln1778-Asp1840 (Lenting, P.J., Donath, M.J.S.H., van Mourik, J.A., and Mertens, K. (1994) J. Biol. Chem. 269, 7150-7155). Employing distinct recombinant factor VIII fragments, we now have localized the epitope of this antibody more precisely between the A3 domain residues Glu1801 and Met1823. Hydropathy analysis indicated that this region is part of a major hydrophilic exosite within the A3 domain. The interaction of factor IXa with this exosite was studied by employing overlapping synthetic peptides encompassing the factor VII region Tyr1786-Ala1834. Factor IXa binding was found to be particularly efficient to peptide corresponding to the factor VIII sequences Lys1804-Lys1818 and Glu1811-Gln1820. The same peptides proved effective in binding antibody CLB-CAg A. Further analysis revealed that peptides Lys1804-Lys1818 and Glu1811-Gln1820 interfere with binding of factor IXa to immobilized factor VIII light chain (Ki approximately 0.2 mM and 0.3 mM, respectively). Moreover, these peptides inhibit factor X activation by factor IXa in the presence of factor VIIIa (Ki approximately 0.2 mM and 0.3 mM, respectively) but not in its absence. Equilibrium binding studies revealed that these two peptides bind to the factor IX zymogen and its activated form, factor IXa, with the same affinity (apparent Kd approximately 0.2 mM), whereas the complete factor VIII light chain displays preferential binding to factor IXa. In conclusion, our results demonstrate that peptides consisting of the factor VIII light chain residues Lys1804-Lys1818 and Glu1811-Gln1820 share a factor IXa binding site that is essential for the assembly of the factor X-activating factor IXa-factor VIIIa complex. We propose that the overlapping sequence Glu1811-Lys1818 comprises the minimal requirements for binding to activated factor IX.

Proprotein convertases are responsible for the endoproteolytic processing of prohormones, neuropeptide precursors, and other proproteins within the constitutive and regulated secretory pathways. Cleavage occurs carboxyl-terminally of basic amino acid motifs, such as RX(K/R)R, RXXR, and (R/K)R. As already available for the other known mammalian members of this enzyme family, we here define structural and functional features of human lymphoma proprotein convertase (LPC). Analysis of expression of recombinant LPC in stably transfected Chinese hamster ovary cells reveals biosynthesis of a 92-kDa nonglycosylated precursor (proLPC) and a 102-kDa endoglycosidase H-sensitive glycosylated form of proLPC. Only the latter is further processed and after propeptide removal converted into a complexly N-glycosylated mature form of LPC of about 92 kDa. Co-expression experiments of truncated LPC with an active site mutant of LPC (LPCS265A) indicate that prodomain removal of LPC occurs via an autoproteolytic, intramolecular mechanism, as was demonstrated before for some of the other members of this enzyme family. Prodomain removal is shown to be required for LPC to exit the endoplasmic reticulum. As far as subcellular localization is concerned, immunocytochemical, ultrastructural, and biochemical analyses show that LPC is concentrated in the trans-Golgi network, associated with membranes, and not secreted. Carboxyl-terminal domains are critically involved in this cellular retention, because removal of both the hydrophobic region and the cytoplasmic tail of LPC results in secretion. Of interest are the observations that LPC is not phosphorylated like furin but is palmitoylated in its cytoplasmic tail. Finally, substrate specificity of LPC is similar to that of furin but not identical. Whereas for furin a basic substrate residue at position P-2 is dispensable, it is essential for LPC. For optimal LPC substrate processing activity, an arginine at position P-6 is preferred over an arginine at P-4.

Lymphoma proprotein convertase (LPC) is a subtilisin-like serine protease of the mammalian proprotein convertase family. It is synthesized as an inactive precursor protein, and propeptide cleavage occurs via intramolecular cleavage in the endoplasmic reticulum. In contrast to other convertases like furin and proprotein convertase-1, propeptide cleavage occurs slowly. Also, both a glycosylated and an unglycosylated precursor are detected. Here we demonstrate that the unglycosylated precursor form of LPC is localized in the cytosol due to the absence of a signal peptide. Using a reducible cross-linker, we found that glycosylated pro-LPC is associated with the molecular chaperone BiP. In addition, we show that pro-LPC is prone to aggregation and forms large complexes linked via interchain disulfide bonds. BiP is associated mainly with non-aggregated pro-LPC and pro-LPC dimers and trimers, suggesting that BiP prevents aggregation. Overexpression of wild-type BiP or a dominant-negative BiP ATPase mutant resulted in reduced processing of pro-LPC. Taken together, these results suggest that binding of BiP to pro-LPC prevents aggregation, but results in slower maturation.

Proprotein convertases are responsible for the endoproteolytic activation of proproteins in the secretory pathway. The most recently discovered member of this family, lymphoma proprotein convertase (LPC), is a type-I transmembrane protein. Previously, we have demonstrated that its cytoplasmic tail is palmitoylated. In this study, we have identified the two most proximal cysteine residues in the cytoplasmic tail as palmitoylation sites. Substitution of either cysteine residue by alanine interfered with palmitoylation of the other. Palmitoylation of LPC was found to be sensitive to the protein palmitoyltransferase inhibitor tunicamycin but not cerulenin. It was also insensitive to the drugs brefeldin A, monensin and cycloheximide, indicating that the modification occurs in a late exocytic or endocytic compartment. Turnover of palmitoylated LPC is significantly faster (t(1/2) approximately 50 min) than that of the LPC polypeptide backbone (t(1/2) approximately 3 h), suggesting that palmitoylation is reversible. Abrogation of palmitoylation reduced the half-life of the LPC protein, but did not affect steady-state localization of LPC in the trans-Golgi network. Finally, LPC could not be detected in detergent-resistant membrane rafts. Taken together, these results suggest that dynamic palmitoylation of LPC is important for stability, but does not function as a dominant trafficking signal.