The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation

Abstract

Seven secretory mammalian kexin-like subtilases have been identified that cleave a variety of precursor proteins at monobasic and dibasic residues. The recently characterized pyrolysin-like subtilase SKI-1 cleaves proproteins at nonbasic residues. In this work we describe the properties of a proteinase K-like subtilase, neural apoptosis-regulated convertase 1 (NARC-1), representing the ninth member of the secretory subtilase family. Biosynthetic and microsequencing analyses of WT and mutant enzyme revealed that human and mouse pro-NARC-1 are autocatalytically and intramolecularly processed into NARC-1 at the (Y,I)VV(V,L)(L,M) downward arrow motif, a site that is representative of its enzymic specificity. In vitro peptide processing studies andor Ala substitutions of the P1-P5 sites suggested that hydrophobicaliphatic residues are more critical at P1, P3, and P5 than at P2 or P4. NARC-1 expression is highest in neuroepithelioma SK-N-MCIXC, hepatic BRL-3A, and in colon carcinoma LoVo-C5 cell lines. In situ hybridization and Northern blot analyses of NARC-1 expression during development in the adult and after partial hepatectomy revealed that it is expressed in cells that have the capacity to proliferate and differentiate. These include hepatocytes, kidney mesenchymal cells, intestinal ileum, and colon epithelia as well as embryonic brain telencephalon neurons. Accordingly, transfection of NARC-1 in primary cultures of embryonic day 13.5 telencephalon cells led to enhanced recruitment of undifferentiated neural progenitor cells into the neuronal lineage, suggesting that NARC-1 is implicated in the differentiation of cortical neurons.

Figure 1

Schematic of NARC-1 and its closest family members. Their catalytic subunits share ≈42% identity (id) and their lengths in amino acids are indicated 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 sites in the prosegment (pro). CT, C-terminal domain.

Figure 2

Biosynthesis of NARC-1 and its mutants. CHO (A) or HK293 (B–F) cells stably or transiently transfected, respectively, with vectors expressing hNARC-1-V5 (A–F) or mNARC-1-V5 (C) were pulse-labeled (P) with [³⁵S]EasyTag Express mix for the indicated time (in min), 4 h (B–E), or 2 h (F). Cell extracts (C) and media (M) were immunoprecipitated with a V5 antibody and the precipitates were resolved by SDS/PAGE on an 8% tricine (A and C–F) or glycine (B) gel. The migration positions of molecular mass standards (kDa), pro-NARC-1 (pN), NARC-1 (N), its secondary site-cleaved product (open arrow), or its pro-segment (pro) are emphasized. (A) Pulse–chase analysis in the absence or presence of brefeldin A (BFA). (B) Effect of tunicamycin (Tun) and the N-glycosylation mutant N533A on NARC-1 processing. (C) Comparison of mNARC-1 and hNARC-1 processing. (D) Processing of WT, P1, P2, P3, P4, and P5 Ala mutants; stars indicate the absence of processing. (E) Processing of the active site mutant H226A. (F) Oligomerization (open triangles) of proNARC-1 in the presence (+) or absence (−) of reducing agent (SH).

Figure 3

In vitro fluorogenic assay of NARC-1 activity. Incubations of 10 μl of 10× concentrated media of HK293 cells expressing hNARC-1, mNARC-1, or empty vector (Ctl): (A) for 3 or 8 h with Suc-RPFHLLVY-MCA, (B) for 8 h with Suc-LLVY-MCA, or (C) for 18 h with Boc-VVVL-MCA. (Upper) The matrix-assisted laser desorption ionization time-of-flight mass spectra of the 8-h (A and B) or 18-h (C) mNARC-1 digests of each substrate. (Lower) The quantitation of released 7-amino-4-methylcoumarin fluorescence from time 0 to the indicated incubation times. Note the formation of RPHFLLVY-OH, Suc-LLVY-OH, and Boc-VVVL-OH as indicated by arrows, demonstrating cleavage N-terminal to MCA.

Figure 4

ISH of NARC-1 during mouse/rat development and in adulthood. (A) ISH of mNARC-1 in mouse whole mounts at E9, E12, E15, and E17 and P3 and P7 by using antisense (AS) or control sense (S) riboprobes. (B) ISH of rNARC-1 in the rat kidney at P6 and P12 and in adulthood. (C) Coronal and sagital analysis of mNARC-1 expression in the adult brain. Li, liver; Ki, kidney; Sk, skin; In, intestine; Ce, cerebellum; Te, telencephalon; and EGL, external granular layer of the cerebellum. [Magnification: (A) E9 = ×3.8, E12 = ×2.1, E15 = ×1.2, E17 = ×0.8, P3 = ×0.7, P7 = ×0.6; (B) P6, P12 = ×2.2, adult = ×0.7; (C) ×1.3.]

Figure 5

Cell, tissue, and post-PHx expression of NARC-1. Northern blot analysis of NARC-1 mRNA in 21 rat, mouse, and human cell lines (A) and 17 rat tissues (B) (Sm, smooth; Sk, skeletal; d, day). The open arrow points to the smaller testicular mRNA. The stars denote an independent experiment for LoVo-C5 cells. (C) mRNA expression of NARC-1, PC7, PC5, SKI-1, PACE4, and furin in intact or sham-operated rat liver compared with that in the remaining tissue after PHx, as estimated by semiquantitative ISH (17). Asterisks indicate the 4- and 2.5-fold up-regulation of PC5 and NARC-1, respectively.

Figure 6

Stimulation of cortical neurogenesis. Primary cultures of E13.5 mouse embryonic neural progenitor cells transfected with plasmids encoding EGFP alone (CTL) or both EGFP and NARC-1 (WT) or its mutant H226A. (A) Western blot analysis before fixation of cell lysates or media previously immunoprecipitated as in Fig. 2. Note the similarity of the migration position of secreted NARC-1 from HK293 (WT-C) and primary cortical cells. pN and N are as in Fig. 2. (B) Double-labeling analysis of the expression of EGFP and either the mitotic protein Ki-67 or the neuronal NeuN cell markers. Results from four different experiments performed in duplicate were quantitated as the percentage of EGFP-positive cells that were also positive for Ki-67 or NeuN expression. Expression of NARC-1, but not its H226A mutant, caused a decrease in the number of mitotic neural progenitor cells (P < 0.005) and an increase in the number of postmitotic neurons (P < 0.01).