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The Secretory Proprotein Convertase Neural Apoptosis-Regulated Convertase 1 (NARC-1): Liver Regeneration and Neuronal Differentiation

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The Secretory Proprotein Convertase Neural Apoptosis-Regulated Convertase 1 (NARC-1): Liver Regeneration and Neuronal Differentiation The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): Liver regeneration and neuronal differentiation Nabil G. Seidah*†, Suzanne Benjannet*, Louise Wickham*, Jadwiga Marcinkiewicz*, Ste´phanie Be´langer Jasmin‡, Stefano Stifani‡, Ajoy Basak§, Annik Prat*, and Michel Chre´ tien§ *Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, QC, H2W 1R7 Canada; ‡Montreal Neurological Institute, McGill University, Montreal, QC, H3A 2B4 Canada; and §Regional Protein Chemistry Center and Diseases of Aging Unit, Ottawa Health Research Institute, Ottawa Hospital, Civic Campus, 725 Parkdale Avenue, Ottawa, ON, K1Y 4E9 Canada Edited by Donald F. Steiner, University of Chicago, Chicago, IL, and approved December 5, 2002 (received for review September 10, 2002) Seven secretory mammalian kexin-like subtilases have been iden- LP251 (Eli Lilly, patent no. WO 02͞14358 A2) recently cloned tified that cleave a variety of precursor proteins at monobasic and by two pharmaceutical companies. NARC-1 was identified via dibasic residues. The recently characterized pyrolysin-like subtilase the cloning of cDNAs up-regulated after apoptosis induced by SKI-1 cleaves proproteins at nonbasic residues. In this work we serum deprivation in primary cerebellar neurons, whereas LP251 describe the properties of a proteinase K-like subtilase, neural was discovered via global cloning of secretory proteins. Aside apoptosis-regulated convertase 1 (NARC-1), representing the ninth from the fact that NARC-1 mRNA is expressed in liver ϾϾ member of the secretory subtilase family. Biosynthetic and micro- testis Ͼ kidney and that the gene localizes to human chromo- sequencing analyses of WT and mutant enzyme revealed that some 1p33-p34.3, no information is available on NARC-1 ac- human and mouse pro-NARC-1 are autocatalytically and intramo- tivity, cleavage specificity, cellular and tissue expression, and lecularly processed into NARC-1 at the (Y,I)VV(V,L)(L,M)2 motif, a biological function. site that is representative of its enzymic specificity. In vitro peptide In this article, we show that NARC-1 belongs to the proteinase processing studies and͞or Ala substitutions of the P1–P5 sites suggested that hydrophobic͞aliphatic residues are more critical at K subfamily of subtilases. It is synthesized as a soluble zymogen that undergoes autocatalytic intramolecular processing in the P1, P3, and P5 than at P2 or P4. NARC-1 expression is highest in 2 neuroepithelioma SK-N-MCIXC, hepatic BRL-3A, and in colon car- endoplasmic reticulum (ER) at the (Y,I)VV(V,L)(L,M) motif, cinoma LoVo-C5 cell lines. In situ hybridization and Northern blot resulting in the cleavage of its prosegment that remains associ- analyses of NARC-1 expression during development in the adult ated with the secreted enzyme. Tissue distribution analysis in and after partial hepatectomy revealed that it is expressed in cells adulthood and during ontogeny in mouse and rat by Northern that have the capacity to proliferate and differentiate. These blots and in situ hybridization histochemistry (ISH) revealed that include hepatocytes, kidney mesenchymal cells, intestinal ileum, NARC-1 mRNA is expressed in a limited number of tissues and colon epithelia as well as embryonic brain telencephalon including the liver, kidney, cerebellum, and small intestine. Its neurons. Accordingly, transfection of NARC-1 in primary cultures of expression is sometimes transient, as in kidney epithelial and embryonic day 13.5 telencephalon cells led to enhanced recruit- brain telencephalon cells. On induction of liver regeneration ment of undifferentiated neural progenitor cells into the neuronal after partial hepatectomy (PHx) in the rat, NARC-1 mRNA lineage, suggesting that NARC-1 is implicated in the differentiation levels peaked on days 2–3, whereas those of PC5 were maximal of cortical neurons. on day 1. NARC-1 overexpression in primary embryonic telen- cephalon cells obtained from embryonic day 13.5 (E13.5) mice cleavage specificity ͉ hypercholesterolemia ͉ neurogenesis ͉ hepatogenesis results in a higher percentage of differentiated neurons. roproteins are the fundamental units from which bioactive Materials and Methods Pproteins and peptides are derived by limited proteolysis. Cloning of Mouse, Rat, and Human NARC-1 and Their Mutants. The Eight of the mammalian proprotein convertases (PCs) respon- GenBank accession nos. of human, mouse, and rat NARC-1 sible for the tissue-specific processing of secretory precursors cDNAs are AX127530, AX207688, and AX207690, respectively. have been identified since 1990. Of these, seven belong to the Full-length human NARC-1 (hNARC-1) (nucleotides 233– yeast kexin subfamily of subtilases and exhibit cleavage- speci- 2311) was cloned by RT-PCR from HepG2 cells, and full-length ficity for basic sites respecting the consensus (K,R)-(X) -(K,R)2, n mouse (nucleotides 137–2259) and partial rat (nucleotides 1215– where n ϭ 0, 2, 4, or 6. They are known as furin, PC1͞3, PC2, 2284) orthologs were obtained from the liver. RT-PCRs were PC4, PACE4, PC5͞6, and PC7͞LPC, respectively (1–3). Al- though less generalized, limited proteolysis also occurs at amino performed by using a Titan (Roche Diagnostics) one-tube kit. acids such as L, V, M, A, T, and S (4, 5). Recently, a subtilisin- The cDNAs were transferred to a derivative of the mammalian kexin-like isozyme called SKI-1͞S1P, belonging to the pyrolysin expression vector pIRES2-EGFP (enhanced GFP) (CLON- subfamily of subtilases was identified (6, 7). It cleaves at nonbasic TECH) that contains a C-terminal V5 epitope, allowing protein residues in the (R,K)-X-(hydrophobic)-(L,T,K,F)2 motif (3, detection with a V5 mAb (Invitrogen) (11, 13). Mutations were 6–11). introduced by PCR, as described (11). Evidence for the existence of processing sites not recognized by the known PCs (12) (M. J. Vincent, personal communication) prompted us to mine databases. By using short, conserved This paper was submitted directly (Track II) to the PNAS office. segments of the SKI-1 catalytic subunit as baits and the protein Abbreviations: EGFP, enhanced GFP; En, embryonic day; ER, endoplasmic reticulum; ISH, ͞ in situ hybridization histochemistry; MCA, 4-methylcoumarin-7-amide; NARC-1, neural BLAST program (www.ncbi.nlm.nih.gov BLAST), we identified apoptosis-regulated convertase 1; hNARC-1, human NARC-1; mNARC-1, mouse NARC-1; Pn, in the patented database a putative convertase called neural postpartum day; PC, proprotein convertase; CHO, Chinese hamster ovary; PHx, partial apoptosis-regulated convertase 1 (NARC-1; Millenium Phar- hepatectomy; RE-OP, rostral extension of the olfactory peduncle. maceuticals, Cambridge, MA, patent no. WO 01͞57081 A2) and †To whom correspondence should be addressed. E-mail: [email protected]. 928–933 ͉ PNAS ͉ February 4, 2003 ͉ vol. 100 ͉ no. 3 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0335507100 Downloaded by guest on September 25, 2021 Biosynthetic Analysis. Recombinant vectors were used to express hNARC-1 and mouse NARC-1 (mNARC-1) in HK293, Chinese hamster ovary (CHO), and Neuro2A cells by transfection with Effectene͞Lipofectamine. Transfected cells were pulse- or pulse–chase-labeled with [35S]EasyTag Express Protein labeling mix (Perkin–Elmer) for various times in the presence or absence of tunicamycin (5 ␮g͞ml) or brefeldin A (2.5 ␮g͞ml). The media and cell lysates were immunoprecipitated with V5 mAb, and the Fig. 1. Schematic of NARC-1 and its closest family members. Their catalytic immune complexes were resolved by SDS͞PAGE on 8% poly- subunits share Ϸ42% identity (id) and their lengths in amino acids are indi- acrylamide͞tricine or glycine gels (13). cated on the right. The positions of the RRG(E,D) and the single N- glycosylation site in the P domain (P dom) are emphasized along with that of the signal peptide (SP) and the primary and putative secondary (?) cleavage In Vitro Enzymatic Assays. Media from HK293 cells expressing sites in the prosegment (pro). CT, C-terminal domain. pIRES2-EGFP alone or recombinant h͞mNARC-1 were con- centrated 10-fold in a Centricon 30 (Millipore). Ten microliters of 10ϫ concentrated media was incubated at 37°C for 3–18 h with Results 50 ␮M of Suc-RPFHLLVY-MCA (4-methylcoumarin-7-amide), Protein Sequence Analysis and Phylogeny of hNARC-1, mNARC-1, and Suc-LLVY-MCA (Enzyme Systems Products, Livermore, CA) or Rat NARC-1. hNARC-1, mNARC-1, and rat NARC-1 (Fig. 1) Boc-VVVL-MCA (A.B., unpublished work) in 25 mM Tris͞Mes, catalytic domains (amino acids 143–473 in hNARC-1) share ϩ pH 7.4 2.5 mM CaCl2 and 0.5% SDS. Fluorescence and 91–92% identity. Using the PILEUP program (GCG), alignment matrix-assisted laser desorption ionization time-of-flight analy- of NARC-1 sequences with those of all other known subtilases sis of the products were as described (14). (23) revealed that this enzyme is a mammalian PC member of the proteinase K subfamily of subtilases (Ϸ42% identity in the Northern Blot and ISH. Northern blot analyses (15) were per- catalytic domain of proteinase T, R, and K). NARC-1 sequences formed on poly(A)ϩ mRNA from cell lines (16) or 3-month-old essentially differ by an N-terminal extension of the rat and mouse Ϸ250-g Sprague–Dawley male rat tissues, using an RNeasy signal peptides (85 and 44 aa compared with 30 aa for hNARC-1) protocol (Qiagen, Chatsworth, CA). The blots were hybridized because of the presence of an upstream in-frame AUG. How- overnight at 68°C with [32P]UTP NARC-1 cRNA probes com- ever, we cannot rule out that the first AUG is not the initiator, prising nucleotides 784–1406, 1197–2090, or 1215–2284 of hu- as reported for the metalloprotease nardilysin (24). Following man, mouse, or rat NARC-1, respectively. For ISH, mouse or rat the signal peptide, hNARC-1 exhibits a putative prosegment preceding the catalytic subunit characterized by the presence of sense and antisense cRNA probes coding for NARC-1, furin, the canonical Asp-186, His-226, and Ser-386 catalytic triad and SKI-1, PC7, PACE4, and PC5 were labeled with 35S-UTP and 35 ͞ the oxyanion hole Asn-317.
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