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 complexlyN-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.

[1]  J. Leunissen,et al.  Subtilases: The superfamily of subtilisin‐like serine proteases , 1997, Protein science : a publication of the Protein Society.

[2]  M. Spence,et al.  Endoprotease Activities Other Than Furin and PACE4 with a Role in Processing of HIV-I gp160 Glycoproteins in CHO-K1 Cells* , 1997, The Journal of Biological Chemistry.

[3]  C Di Bello,et al.  Identification of the Paired Basic Convertases Implicated in HIV gp160 Processing Based on in Vitro Assays and Expression in CD4+ Cell Lines* , 1996, The Journal of Biological Chemistry.

[4]  G. Warren,et al.  The role of the membrane-spanning domain and stalk region of N-acetylglucosaminyltransferase I in retention, kin recognition and structural maintenance of the Golgi apparatus in HeLa cells. , 1996, Journal of cell science.

[5]  D. Constam,et al.  SPC4, SPC6, and the novel protease SPC7 are coexpressed with bone morphogenetic proteins at distinct sites during embryogenesis , 1996, The Journal of cell biology.

[6]  R. Darnell,et al.  A novel adaptor-related protein complex , 1996, The Journal of cell biology.

[7]  N. Seidah,et al.  cDNA structure, tissue distribution, and chromosomal localization of rat PC7, a novel mammalian proprotein convertase closest to yeast kexin-like proteinases. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[8]  T. Martin,et al.  PC8 [corrected], a new member of the convertase family. , 1996, The Biochemical journal.

[9]  T. Lister,et al.  A new member of the proprotein convertase gene family (LPC) is located at a chromosome translocation breakpoint in lymphomas. , 1996, Cancer research.

[10]  H. Munier-Lehmann,et al.  A Casein Kinase II Phosphorylation Site in the Cytoplasmic Domain of the Cation-dependent Mannose 6-Phosphate Receptor Determines the High Affinity Interaction of the AP-1 Golgi Assembly Proteins with Membranes (*) , 1996, The Journal of Biological Chemistry.

[11]  G. Thomas,et al.  Intracellular trafficking of furin is modulated by the phosphorylation state of a casein kinase II site in its cytoplasmic tail. , 1995, The EMBO journal.

[12]  K. Nakayama,et al.  Localization of Furin to the trans-Golgi Network and Recycling from the Cell Surface Involves Ser and Tyr Residues within the Cytoplasmic Domain (*) , 1995, The Journal of Biological Chemistry.

[13]  J. Bonifacino,et al.  An acidic sequence within the cytoplasmic domain of furin functions as a determinant of trans‐Golgi network localization and internalization from the cell surface. , 1995, The EMBO journal.

[14]  W. V. D. Van de Ven,et al.  Neuroendocrine-specific Expression of the Human Prohormone Convertase 1 Gene , 1995, The Journal of Biological Chemistry.

[15]  M. Vey,et al.  Two independent targeting signals in the cytoplasmic domain determine trans‐Golgi network localization and endosomal trafficking of the proprotein convertase furin. , 1995, The EMBO journal.

[16]  G. Milligan,et al.  The dynamic role of palmitoylation in signal transduction. , 1995, Trends in biochemical sciences.

[17]  Marian C. Horzinek,et al.  Oligomerization of a trans-Golgi/ trans-Golgi Network Retained Protein Occurs in the Golgi Complex and May Be Part of Its Retention (*) , 1995, The Journal of Biological Chemistry.

[18]  H. Klenk,et al.  Endoproteolytic Cleavage of Its Propeptide Is a Prerequisite for Efficient Transport of Furin Out of the Endoplasmic Reticulum (*) , 1995, The Journal of Biological Chemistry.

[19]  H. Klenk,et al.  Maturation of the trans-Golgi network protease furin: compartmentalization of propeptide removal, substrate cleavage, and COOH-terminal truncation , 1994, The Journal of cell biology.

[20]  J. Bonifacino,et al.  The cytoplasmic domain mediates localization of furin to the trans- Golgi network en route to the endosomal/lysosomal system , 1994, The Journal of cell biology.

[21]  T. Watanabe,et al.  Sequence requirements for endoproteolytic processing of precursor proteins by furin: transfection and in vitro experiments. , 1994, Journal of Biochemistry (Tokyo).

[22]  R. Siezen,et al.  Homology modelling of the catalytic domain of human furin. A model for the eukaryotic subtilisin-like proprotein convertases. , 1994, European journal of biochemistry.

[23]  M. Hamaguchi,et al.  A furin-defective cell line is able to process correctly the gp160 of human immunodeficiency virus type 1 , 1994, Journal of virology.

[24]  A. Roebroek,et al.  Expression of the dibasic proprotein processing enzyme furin is directed by multiple promoters. , 1994, The Journal of biological chemistry.

[25]  G. Thomas,et al.  Intracellular trafficking and activation of the furin proprotein convertase: localization to the TGN and recycling from the cell surface. , 1994, The EMBO journal.

[26]  N. Seidah,et al.  The family of subtilisin/kexin like pro-protein and pro-hormone convertases: divergent or shared functions. , 1994, Biochimie.

[27]  A. Rehemtulla,et al.  PACE4 is a member of the mammalian propeptidase family that has overlapping but not identical substrate specificity to PACE. , 1993, Biochemistry.

[28]  S. Wong,et al.  The SXYQRL sequence in the cytoplasmic domain of TGN38 plays a major role in trans-Golgi network localization. , 1993, The Journal of biological chemistry.

[29]  R. Siezen,et al.  Modulation of furin-mediated proprotein processing activity by site-directed mutagenesis. , 1993, The Journal of biological chemistry.

[30]  Dr. Gareth Griffiths.  Fine Structure Immunocytochemistry , 1993, Springer Berlin Heidelberg.

[31]  Van de Ven Wj,et al.  Structure and function of eukaryotic proprotein processing enzymes of the subtilisin family of serine proteases. , 1993 .

[32]  R. Wright,et al.  Mutation of a tyrosine localization signal in the cytosolic tail of yeast Kex2 protease disrupts Golgi retention and results in default transport to the vacuole. , 1992, Molecular biology of the cell.

[33]  R. Leduc,et al.  Activation of human furin precursor processing endoprotease occurs by an intramolecular autoproteolytic cleavage. , 1992, The Journal of biological chemistry.

[34]  R. Klausner,et al.  A novel di-leucine motif and a tyrosine-based motif independently mediate lysosomal targeting and endocytosis of CD3 chains , 1992, Cell.

[35]  R. Espinosa,et al.  Identification and analysis of the gene encoding human PC2, a prohormone convertase expressed in neuroendocrine tissues. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[36]  T. Kreis,et al.  Recruitment of coat proteins onto Golgi membranes in intact and permeabilized cells: Effects of brefeldin A and G protein activators , 1992, Cell.

[37]  A. V. D. van den Ouweland,et al.  Development and characterization of a panel of monoclonal antibodies against the novel subtilisin-like proprotein processing enzyme furin. , 1992, Hybridoma.

[38]  C. A. Wilcox,et al.  Posttranslational processing of the prohormone-cleaving Kex2 protease in the Saccharomyces cerevisiae secretory pathway , 1991, The Journal of cell biology.

[39]  T. Watanabe,et al.  Arg-X-Lys/Arg-Arg motif as a signal for precursor cleavage catalyzed by furin within the constitutive secretory pathway. , 1991, The Journal of biological chemistry.

[40]  K. Redding,et al.  Immunolocalization of Kex2 protease identifies a putative late Golgi compartment in the yeast Saccharomyces cerevisiae , 1991, The Journal of cell biology.

[41]  J. Slot,et al.  Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat , 1991, The Journal of cell biology.

[42]  A. Brake,et al.  Expression of a human proprotein processing enzyme: correct cleavage of the von Willebrand factor precursor at a paired basic amino acid site. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[43]  J. Hutton.  Subtilisin-like proteinases involved in the activation of proproteins of the eukaryotic secretory pathway. , 1990, Current opinion in cell biology.

[44]  K. Howell,et al.  Identification, sequencing and expression of an integral membrane protein of the trans-Golgi network (TGN38). , 1990, The Biochemical journal.

[45]  A. V. D. van den Ouweland,et al.  Structural homology between the human fur gene product and the subtilisin-like protease encoded by yeast KEX2. , 1990, Nucleic acids research.

[46]  J. Thorner,et al.  Intracellular targeting and structural conservation of a prohormone-processing endoprotease. , 1989, Science.

[47]  J. Thorner,et al.  Enzymes required for yeast prohormone processing. , 1988, Annual review of physiology.

[48]  G. Heijne.  A new method for predicting signal sequence cleavage sites. , 1986 .

[49]  Y. Fujiki,et al.  Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum , 1982, The Journal of cell biology.