Biosynthesis, distinct post-translational modifications, and functional characterization of lymphoma proprotein convertase.
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.
van de Loo
J W
JW
Laboratory for Molecular Oncology, Center for Human Genetics, University of Leuven and Flanders Interuniversity Institute for Biotechnology, Herestraat 49, 3000 Leuven, Belgium.
Creemers
J W
JW
Bright
N A
NA
Young
B D
BD
Roebroek
A J
AJ
Van de Ven
W J
WJ
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D019556:COS Cells; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D006224:Cricetinae; D019084:Fluorescent Antibody Technique, Indirect; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology / Q000648:ultrastructure; D006801:Humans; D008223:Lymphoma / Q000201:enzymology*; D008322:Mammals; D016253:Microscopy, Immunoelectron; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid / Q000378:metabolism; D017354:Point Mutation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000378:metabolism*; D013381:Subtilisins; D014162:Transfection; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D019308:Palmitic Acid; C099101:PCSK7 protein, human; D012697:Serine Endopeptidases; D013381:Subtilisins