N-arginine dibasic convertase, a metalloendopeptidase as a prototype of a class of processing enzymes.

Abstract

N-Arg dibasic convertase is a me tidase from rat brain cortex and testis that cleaves peptide substrates on the N terminu ofArg residues in dibasic s s.By using both an oinu ide and antibodies to screen a rat testis cDNA library, a fill-length cDNA (4,5). On the basis of its similarity to subtilisin and to furin (6), a human homolog of the Kex2 protein, a family of prohormone convertases (PCs) has been identified by PCR techniques. Their involvement in processing of a number of propeptides and proproteins was inferred mainly from cotransfection experiments (7-9), and for PC1, by the use of antisense mRNA (10).Characterization of putative processing endoproteases by classical biochemical techniques has led to the identification of a number of activities, selective for basic residues in precursors, that belong to the four classes of proteases (metallo-, serine, aspartyl, and thiol enzymes; for review, see ref. 11). This suggested that more than one processing endoprotease family could exist (12). To our knowledge, none of these basic-residue-specific enzymes has been cloned.Recently, a metalloendopeptidase was completely purified from rat testis and shown to cleave a number of peptide substrates on the N terminus of Arg residues in dibasic moieties (13). This enzyme was also present in rat brain cortex and its functional properties appeared undistinguishable from those of the somatostatin-28 convertase activity previously identified in this tissue (14,15). By using microsequencing of tryptic fragments of the purified enzyme to design an oligonucleotide probe and polyclonal antibodies raised against the purified protein (13) (20)].The in vitro highly restricted specificity ofNRD convertase for Arg residues in dibasic processing signals and its belonging to the M16 family, which contains other enzymes involved in maturation, suggest that this enzyme is the prototype of a distinct family of processing endoproteases. MATERIALS AND METHODSIsolatin and Cherizatin of cDNA Cones E d NRD Convertae. Four tryptic fragments were sequenced after digestion of previously purified NRD convertase following native PAGE. One fragment, GMQLIYLPPSPLLAE, was used to design the following degenerate inosine-containing oligonucleotide: 5'-GGIGGIAG(A/G)TAIATIA(G/A)(T/ C)TGCATICC-3'. Two additional peptides were obtained by endolysine C treatment (13) autoradiography of the filters, positive phage plaques were isolated and rescreened to obtain single purified phage isolates. A similar aliquot ofthe library was plated onto NZ-amine medium plates for screening using polyclonal antibodies raised Abbreviations: NRD convertase, N-arginine dibasic convertase; IDE, insulin degrading enzyme; MPP, mitochondrial matrixprocessing peptidase.

Distinct Properties of Neuronal and Astrocytic Endopeptidase 3.4.24.16: A Study on Differentiation, Subcellular Distribution, and Secretion Processes

“…Second, we detected two stretches of charged residues in the middle of the endopeptidase 3.4.24.16 sequence. Interestingly, a similar cluster of charged residues was found in a proteolytic activity, the N-arginine dibasic convertase (Pierotti et al, 1994), as well as in two other types of proteins: carboxypeptidase E (CPE), where this type of sequence (Fricker et al, 1986) acts as an amphiphilic helix responsible for the attachment of CPE to the membrane (Mitra et al, 1994), and complexins in which comparable stretches of hydrophilic residues have been implicated in their interaction with the SNAP-receptor core complex (McMahon et al, 1995). Altogether, these observations are consistent with the possibility that endopeptidase 3.4.24.16 interacts with proteins able to target the enzyme to the membrane via its hydrophilic sequence.…”

Ahi-1 , a Novel Gene Encoding a Modular Protein with WD40-Repeat and SH3 Domains, Is Targeted by the Ahi-1 and Mis-2 Provirus Integrations

“…3A). Such a domain is found in quite different proteins, such as nucleolines, calcium-binding proteins, and transcription factors (40). Finally, three potential PEST sequences (at residues 19 to 30, 100 to 118, and 417 to 432) (46), three potential nuclear localization signal (aa 62 to 94, 134 to 151, and 825 to 868) (10), and four potential glycosylation sites (at positions 310, 536, 821, and 851) were found in the Ahi-1 protein.…”

Aminopeptidase B from the rat testis is a bifunctional enzyme structurally related to leukotriene-A4 hydrolase

“…However, complete removal of the basic residues of a doublet in a precursor can, in principle, proceed alternatively by three different pathways, of which two may involve an aminopeptidase-B (Ap-B) (1). Several reports showed the existence of endoproteases with a cleavage specificity for the N terminus of basic residues present in the doublet (8)(9)(10)(11)(12) and of poorly characterized Ap-B-like activities (9,(13)(14)(15)). An Ap-B (EC 3.4.11.6) activity initially was identified in several rat tissues (13) and was defined as an exopeptidase able to remove basic residues from the N terminus of L-amino acid-␤-naphthylamide, dipeptides, and kallidin 10 (13,14).…”