Asparagine-linked oligosaccharide-independent secretion of egg envelope glycoprotein ZPC of the Japanese quail (Coturnix japonica)

In avian species, a glycoprotein homologous to mammalian ZPC is synthesized in the granulosa cells of developing follicles. We have previously reported that the newly synthesized ZPC (proZPC) in the granulosa cells is cleaved at the consensus furin cleavage site to generate mature ZPC prior to secretion. In the present study, we examined the role of asparagine (N)-linked oligosaccharides in the proteolytic processing of proZPC and the subsequent secretion of ZPC by using site-directed mutagenesis of the consensus sequence for N-glycosylation, and tunicamycin, an inhibitor for N-glycosylation of glycoprotein. Western blot analysis demonstrated that tunicamycin did not block either proteolytic cleavage of proZPC or the subsequent ZPC secretion. Moreover, a site-directed mutant that possesses a mutated sequence for N-glycosylation was efficiently secreted from the cells. These results indicate that proteolytic cleavage of proZPC, and the subsequent ZPC secretion occur in the absence of N-linked oligosaccharides. Therefore, the addition of N-glycans to ZPC polypeptide is not required for quail ZPC secretion.     

Secretion of egg envelope protein ZPC after C-terminal proteolytic processing in quail granulosa cells.

In avian species, an egg envelope homologous to the mammalian zona pellucida is called the perivitelline membrane. We have previously reported that one of its components, a glycoprotein homologous to mammalian ZPC, is synthesized in the granulosa cells of the quail ovary. In the present study, we investigated the proteolytic cleavage of the newly synthesized ZPC and the secretion of ZPC from the granulosa cells. Western blot analysis of the cell lysates demonstrated that the 43-kDa protein is the precursor of mature ZPC (proZPC), and is converted to the 35-kDa protein before secretion. The accumulation of proZPC in the presence of brefeldin A, and conversion of proZPC to ZPC in the presence of monensin, indicate the possibility that the proteolytic processing of ZPC occurs in the Golgi apparatus. An analysis of amino-acid sequence identified that the C terminus of mature ZPC protein is Phe360, and the N-terminal amino-acid sequence of the proZPC-derived fragment was determined as Asp363. These results suggest that newly synthesized ZPC is cleaved at the consensus furin cleavage site, and the resulting two basic residues at the C terminus are subsequently trimmed off to generate mature ZPC prior to secretion.     

Variant of perivitelline membrane glycoprotein ZPC of Japanese quail (Coturnix japonica) lacking its cytoplasmic tail exhibits the retention in the endoplasmic reticulum of Chinese hamster ovary (CHO-K1) cells

Avian perivitelline membrane, an investment homologous to the mammalian zona pellucida, is composed of at least two glycoproteins. Our previous studies demonstrated that one of its components, ZPC, which is synthesized in the ovarian granulosa cells, is secreted after carboxy-terminal proteolytic processing, and this event is a prerequisite event for ZPC secretion in quail. In the present study, we examined the role of the cytoplasmic tail, which is successfully removed after proteolytic processing, in membrane transport, proteolytic processing, and the secretion of quail ZPC. In pursuit of this, we produced a truncated ZPC mutant lacking the cytoplasmic tail located in its C-terminus and examined its expression in the mammalian cell line. Western blot analyses demonstrated that the cytoplasmic tail-deficient ZPC was neither secreted nor underwent proteolytic processing in the cells. Immunofluorescence analysis and the acquisition of resistance to endoglycosidase H digestion of the cytoplasmic tail-deficient ZPC demonstrated that the deletion of the cytoplasmic tail interferes with the intracellular trafficking of the protein from the endoplasmic reticulum to the Golgi apparatus. These results indicate that the cytoplasmic tail of quail ZPC might possess the determinant responsible for the efficient transport of the newly synthesized ZPC from the endoplasmic reticulum to the Golgi apparatus.     

Furin directly cleaves proMMP-2 in the trans-Golgi network resulting in a nonfunctioning proteinase

Proprotein convertases play an important role in tumorigenesis and invasiveness. Here, we report that a dibasic amino acid convertase, furin, directly cleaves proMMP-2 within the trans-Golgi network leading to an inactive form of matrix metalloproteinase-2 (MMP-2). Co-transfection of COS-1 cells with both proMMP-2 and furin cDNAs resulted in the cleavage of the N-terminal propeptide of proMMP-2. The molecular mass of cleaved MMP-2 (63 kDa), detected in both cell lysates and conditioned medium, is between the intermediate and fully activated forms of MMP-2 induced by membrane type 1-MMP. Furin-cleaved MMP-2 does not possess proteolytic activity as examined in a cell-free assay. Treatment of transfected cells with a furin inhibitor resulted in a dose-dependent inhibition of proMMP-2 cleavage; recombinant tissue inhibitor of metalloproteinase-2, which binds to the active site of membrane type 1-MMP, had no inhibitory effect. Site-directed mutagenesis of amino acids in the furin consensus recognition motif of proMMP-2(R69KPR72) prevented propeptide cleavage, thereby identifying the scissile bond and characterizing the basic amino acids required for cleavage. Other experimental observations were consistent with intracellular furin cleavage of proMMP-2 in the trans-Golgi network. The furin cleavage site in other proMMPs was examined. MMP-3, which contains the RXXR furin consensus sequence, was cleaved in furin co-transfected cells, whereas MMP-1, which lacks an RXXR consensus sequence, was not cleaved. In conclusion, we report the novel observation that furin can directly cleave the RXXR amino acid sequence in the propeptide domain of proMMP-2 leading to inactivation of the enzyme.     

Cleavage of the ADAMTS13 propeptide is not required for protease activity

ADAMTS13 belongs to the "a disintegrin and metalloprotease with thrombospondin repeats" family, and cleaves von Willebrand factor multimers into smaller forms. For several related proteases, normal folding and enzymatic latency depend on an NH2-terminal propeptide that is removed by proteolytic processing during biosynthesis. However, the ADAMTS13 propeptide is unusually short and poorly conserved, suggesting it may not perform these functions. ADAMTS13 was secreted from transfected HeLa cells with a half-time of 7 h and the rate-limiting step was exported from the endoplasmic reticulum. Deletion of the propeptide did not impair the secretion of active ADAMTS13, indicating that the propeptide is dispensable for folding. Furin was shown to be sufficient for ADAMTS13 propeptide processing in two ways. First, mutation of the furin consensus recognition site prevented propeptide cleavage in HeLa cells and resulted in secretion of pro-ADAMTS13. Second, furin-deficient LoVo cells secreted ADAMTS13 with the propeptide intact, and cotransfection with furin restored propeptide cleavage. In both cell lines, secreted pro-ADAMTS13 had normal proteolytic activity toward von Willebrand factor. In cells coexpressing both ADAMTS13 and von Willebrand factor, pro-ADAMTS13 cleaved pro-von Willebrand factor intracellularly. Therefore, the ADAMTS13 propeptide is not required for folding or secretion, and does not perform the common function of maintaining enzyme latency.     

Regulation of bone morphogenetic protein-4 activity by sequence elements within the prodomain

Bone morphogenetic protein-4 (BMP-4) is synthesized as a large precursor protein, which undergoes proprotein convertase-mediated proteolytic maturation along the secretory pathway to release the active ligand. Pro-BMP-4 is initially cleaved at a consensus furin motif adjacent to the mature ligand domain (the S1 site), and this allows for subsequent cleavage at an upstream motif (the S2 site). This sequential cleavage liberates a small, evolutionarily conserved, prodomain fragment (the linker peptide) of unknown fate and function. Here we show that the linker domain is essential for proper folding, exit from the endoplasmic reticulum, and thus cleavage of the BMP-4 precursor when overexpressed in Xenopus oocytes and embryos but not in cultured mammalian cells. Mature BMP-4 synthesized from a precursor in which the S1 site is non-cleavable, such that the linker domain remains covalently attached to the ligand, has little or no activity in vivo. Finally, analysis of folding, cleavage, and bioactivity of chimeric precursors containing the BMP-7 prodomain and BMP-4 mature domain, or vice versa, with or without the BMP-4 linker domain revealed that the linker domain is only functional in the context of the BMP-4 prodomain, and that differential cleavage around this domain can regulate the activity of a heterologous ligand.     

Activation of human furin precursor processing endoprotease occurs by an intramolecular autoproteolytic cleavage.

Human furin is a calcium-dependent serine endoprotease that can efficiently cleave many precursor proteins on the carboxyl side of the consensus cleavage sequence, -Arg-X-Lys/Arg-Arg-, both in vivo and in vitro. Analysis of furin proteins in extracts of cells infected with a vaccinia recombinant expressing human furin show that the enzyme is present as two prominent forms of 90 and 96 kDa. Because the structurally related bacterial subtilisins require endoproteolytic removal of the NH2-terminal pro-region by an autocatalytic intramolecular cleavage, we speculated that the size heterogeneity in the furin doublet similarly may result from a proteolytic removal of an NH2-terminal pro-region. Here we report identification of the 90-kDa furin NH2 terminus and, based on the reported sequence of the furin cDNA, demonstrate that this furin protein is derived from a larger precursor by an endoproteolytic cleavage on the COOH-terminal side of a consensus furin cleavage site, -Arg-Thr-Lys-Arg107-. Expression of mutant furin molecules containing an altered cleavage site (Arg104----Ala or Arg107----Gly) resulted in the production of only the 96-kDa furin protein. Assays of furin-dependent cleavage of a protein substrate in vitro showed that proteolytic activity was associated with the 90-kDa and not the 96-kDa furin protein, demonstrating that removal of the NH2-terminal pro-region is required for furin activity. Expression of a third furin construct containing a mutation of the active site aspartate (Asp153----Asn) similarly resulted in the expression of only the 96-kDa protein, suggesting that furin activation occurs by an autoproteolytic cleavage. Finally, the production of 90-kDa furin from either site-directed furin mutant could not be potentiated by overexpressing active furin, suggesting that the autoproteolytic activation was an intramolecular event.     

Secretion of mouse ZP3, the sperm receptor, requires cleavage of its polypeptide at a consensus furin cleavage-site

The mouse egg extracellular coat, or zona pellucida, consists of three glycoproteins, called mZP1-3. Each glycoprotein possesses a consensus sequence recognized by the furin family of proprotein convertases. Previously, it was reported that mZP2 and mZP3 are cleaved at their consensus furin cleavage-sites located near the C-terminus of the polypeptides [Litscher, E. S., Qi, H., and Wassarman, P. M. (1999) Biochemistry 38, 12280-12287]. Here, use of site-directed mutagenesis of the mZP3 gene and a specific inhibitor of furin-like enzymes revealed that secretion of nascent mZP3 from transfected cells is dependent on cleavage of mZP3 at its consensus furin cleavage-site. The dependence of secretion on cleavage represents a novel function for furin family enzymes.     

Proteolytic activation of respiratory syncytial virus fusion protein. Cleavage at two furin consensus sequences.

The F (fusion) protein of the respiratory syncytial viruses is synthesized as an inactive precursor F(0) that is proteolytically processed at the multibasic sequence KKRKRR(136) into the subunits F(1) and F(2) by the cellular protease furin. This maturation process is essential for the F protein to gain fusion competence. We observed that proteolytic cleavage additionally occurs at another basic motif, RARR(109), that also meets the requirements for furin recognition. Cleavage at both sites leads to the removal from the polypeptide chain of a glycosylated peptide of 27 amino acids. When the sequence RARR(109) was changed to NANR(109) or to RANN(109) by site-directed mutagenesis, cleavage by furin was completely prevented. Although the mutants were still processed at position Arg(136), they did not show any syncytia formation. Proteolytic cleavage of the modified motifs was achieved by treatment of transfected cells with trypsin converting the F mutants into their fusogenic forms. Our findings indicate that both furin consensus sequences have to be cleaved in order to activate the fusion protein.     

Proprotein Convertases Process and Thereby Inactivate Formylglycine-generating Enzyme

Formylglycine-generating enzyme (FGE) post-translationally converts a specific cysteine in newly synthesized sulfatases to formylglycine (FGly). FGly is the key catalytic residue of the sulfatase family, comprising 17 nonredundant enzymes in human that play essential roles in development and homeostasis. FGE, a resident protein of the endoplasmic reticulum, is also secreted. A major fraction of secreted FGE is N-terminally truncated, lacking residues 34–72. Here we demonstrate that this truncated form is generated intracellularly by limited proteolysis mediated by proprotein convertase(s) (PCs) along the secretory pathway. The cleavage site is represented by the sequence RYSR⁷²↓, a motif that is conserved in higher eukaryotic FGEs, implying important functionality. Residues Arg-69 and Arg-72 are critical because their mutation abolishes FGE processing. Furthermore, residues Tyr-70 and Ser-71 confer an unusual property to the cleavage motif such that endogenous as well as overexpressed FGE is only partially processed. FGE is cleaved by furin, PACE4, and PC5a. Processing is disabled in furin-deficient cells but fully restored upon transient furin expression, indicating that furin is the major protease cleaving FGE. Processing by endogenous furin occurs mostly intracellularly, although also extracellular processing is observed in HEK293 cells. Interestingly, the truncated form of secreted FGE no longer possesses FGly-generating activity, whereas the unprocessed form of secreted FGE is active. As always both forms are secreted, we postulate that furin-mediated processing of FGE during secretion is a physiological means of higher eukaryotic cells to regulate FGE activity upon exit from the endoplasmic reticulum.     

Biosynthesis and secretion of insect lipoprotein: involvement of furin in cleavage of the apoB homolog, apolipophorin-II/I

The biosynthesis of neutral fat-transporting lipoproteins involves the lipidation of their nonexchangeable apolipoprotein. In contrast to its mammalian homolog apolipoprotein B, however, insect apolipophorin-II/I (apoLp-II/I) is cleaved posttranslationally at a consensus substrate sequence for furin, resulting in the appearance of two apolipoproteins in insect lipoprotein. To characterize the cleavage process, a truncated cDNA encoding the N-terminal 38% of Locusta migratoria apoLp-II/I, including the cleavage site, was expressed in insect Sf9 cells. This resulted in the secretion of correctly processed apoLp-II and truncated apoLp-I. The cleavage could be impaired by the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (decRVKRcmk) as well as by mutagenesis of the consensus substrate sequence for furin, as indicated by the secretion of uncleaved apoLp-II/I-38. Treatment of L. migratoria fat body, the physiological site of lipoprotein biosynthesis, with decRVKRcmk similarly resulted in the secretion of uncleaved apoLp-II/I, which was integrated in lipoprotein particles of buoyant density identical to wild-type high density lipophorin (HDLp). These results show that apoLp-II/I is posttranslationally cleaved by an insect furin and that biosynthesis and secretion of HDLp can occur independent of this processing step. Structure modeling indicates that the cleavage of apoLp-II/I represents a molecular adaptation in homologous apolipoprotein structures. We propose that cleavage enables specific features of insect lipoproteins, such as low density lipoprotein formation, endocytic recycling, and involvement in coagulation.     

Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen.

Previous work demonstrated that human furin is a predominantly Golgi membrane-localized endoprotease that can efficiently process precursor proteins at paired basic residues (-Lys-Arg- or -Arg-Arg-) in transfected cells. Anion-exchange chromatography of culture supernatant from cells expressing a soluble truncated form of human furin resulted in a greatly enriched preparation of the endoprotease (approximately 70% pure as determined by protein staining). Enzymatic studies show that furin is a calcium-dependent (K0.5 = 200 microM) serine endoprotease which has greater than 50% of maximal activity between pH 6.0 and 8.5. The inhibitor sensitivity of furin suggests that it is similar to, yet distinct from, other calcium-dependent proteases. Evidence that furin may require a P4 Arg in fluorogenic peptide substrates suggested that this enzyme might cleave the protective antigen (PA) component of anthrax toxin at the sequence -Arg-Lys-Lys-Arg-. Indeed, PA was cleaved by purified furin at the proposed consensus site (-Arg-X-Lys/Arg-Arg decreases-) at a rate (8 mumol/min/mg total protein) 400-fold higher than that observed with synthetic peptides. In addition, the processing of mutant PA molecules with altered cleavage sites suggests that furin-catalyzed endoproteolysis minimally requires an -Arg-X-X-Arg- recognition sequence for efficient cleavage. Together, these results support the hypothesis that furin processes protein precursors containing this cleavage site motif in the exocytic pathway and in addition, raises the possibility that the enzyme also cleaves extracellular substrates, including PA.     

Proteolytic cleavage of the ectodomain of the L1 CAM family member Tractin

Tractin is a member of the L1 family of cell adhesion molecules in leech. Immunoblot analysis suggests that Tractin is constitutively cleaved in vivo at a proteolytic site with the sequence RKRRSR. This sequence conforms to the consensus sequence for cleavage by members of the furin family of convertases, and this proteolytic site is shared by a majority of other L1 family members. We provide evidence with furin-specific inhibitor experiments, by site-specific mutagenesis of Tractin constructs expressed in S2 cells, as well as by Tractin expression in furin-deficient LoVo cells that a furin convertase is the likely protease mediating this processing. Cross-immunoprecipitations with Tractin domain-specific antibodies suggest that the resulting NH(2)- and COOH-terminal cleavage fragments interact with each other and that this interaction provides a means for the NH(2)-terminal fragment to be tethered to the membrane. Furthermore, in S2 cell aggregation assays we show that the NH(2)-terminal fragment is necessary for homophilic adhesion and that cells expressing only the transmembrane COOH-terminal fragment are non-adhesive. However, tethering of exogeneously provided Tractin NH(2)-terminal fragment to S2 cells expressing only the COOH-terminal fragment can functionally restore the adhesive properties of Tractin.     

Biologically active APRIL is secreted following intracellular processing in the Golgi apparatus by furin convertase

Tumor necrosis factor (TNF) ligand family members are synthesized as transmembrane proteins, and cleavage of the membrane-anchored proteins from the cell surface is frequently observed. The TNF-related ligands APRIL and BLyS and their cognate receptors BCMA/TACI form a two ligand/two receptor system that has been shown to participate in B- and T-cell stimulation. In contrast to BLyS, which is known to be cleaved from the cell surface, we found that APRIL is processed intracellularly by furin convertase. Blockage of protein transport from the endoplasmic reticulum to the Golgi apparatus by Brefeldin A treatment abrogated APRIL processing, whereas monensin, an inhibitor of post-Golgi transport, did not interfere with cleavage of APRIL, but blocked secretion of processed APRIL. Thus, APRIL shows a unique maturation pathway among the TNF ligand family members, as it not detectable as a membrane-anchored protein at the cell surface, but is processed in the Golgi apparatus prior to its secretion.     

Inhibition of furin by serpin Spn4A from Drosophila melanogaster

The serpin gene Spn4 from Drosophila melanogaster encodes multiple isoforms with alternative reactive site loops (RSL). Here, we show that isoform Spn4A inhibits human furin with an apparent kassoc of 5.5 x 10(6) M(-1) s(-1). The serpin forms SDS-stable complexes with the enzyme and the RSL of Spn4A is cleaved C-terminally to the sequence -Arg-Arg-Lys-Arg/ in accord with the recognition/cleavage site of furin. Immunofluorescence studies show that Spn4A is localized in the endoplasmic reticulum (ER), suggesting that the inhibitor is an interesting tool for investigating the cellular mechanisms regulating furin and for the design of agents controlling prohormone convertases.     

Mouse zona pellucida glycoproteins mZP2 and mZP3 undergo carboxy-terminal proteolytic processing in growing oocytes.

The extracellular coat, or zona pellucida, of the mouse egg consists of three glycoproteins, called mZP1-3. The glycoproteins are synthesized and secreted concomitantly by growing oocytes during their 2-3-week growth phase. Each of the glycoproteins has a consensus furin cleavage site (-Arg-X-Lys/Arg-Arg-) near the C-terminus of their polypeptide. Here, several approaches were employed to determine whether nascent mZP2 and mZP3 are cleaved at the consensus sites, -Arg-Ser-Lys-Arg- and -Arg-Asn-Arg-Arg-, respectively, prior to secretion. Molecular mass determinations of deglycosylated mZP2 and mZP3 suggest that their polypeptides are approximately 9 and approximately 7 kDa smaller, respectively, than predicted from exon sequences. Two-dimensional thin-layer chromatographic analyses were also carried out to identify amino acids released from the C-terminus of mZP2 and mZP3 by carboxypeptidase B. On the basis of exon sequences, there are no Arg residues at the predicted C-terminus of the mature glycoproteins. However, for both mZP2 and mZP3, Arg residues were released by carboxypeptidase B, consistent with processing at the consensus furin cleavage site. Furthermore, an antiserum raised against an mZP3 peptide, located downstream of the consensus furin cleavage site, failed to label purified mZP3 on Western immunoblots. The antiserum also failed to label the zona pellucida of oocytes examined by laser scanning confocal microscopy. Collectively, these results strongly suggest that mZP2 and mZP3 are processed at their consensus furin cleavage site prior to secretion and incorporation into the zona pellucida.     

Template-assisted rational design of peptide inhibitors of furin using the lysine fragment of the mung bean trypsin inhibitor

Highly active, small-molecule furin inhibitors are attractive drug candidates to fend off bacterial exotoxins and viral infection. Based on the 22-residue, active Lys fragment of the mung bean trypsin inhibitor, a series of furin inhibitors were designed and synthesized, and their inhibitory activity towards furin and kexin was evaluated using enzyme kinetic analysis. The most potent inhibitor, containing 16 amino acid residues with a Ki value of 2.45x10(-9) m for furin and of 5.60x10(-7) m for kexin, was designed with three incremental approaches. First, two nonessential Cys residues in the Lys fragment were deleted via a Cys-to-Ser mutation to minimize peptide misfolding. Second, residues in the reactive site of the inhibitor were replaced by the consensus substrate recognition sequence of furin, namely, Arg at P1, Lys at P2, Arg at P4 and Arg at P6. In addition, the P7 residue Asp was substituted with Ala to avoid possible electrostatic interference with furin inhibition. Finally, the extra N-terminal and C-terminal residues beyond the doubly conjugated disulfide loops were further truncated. However, all resultant synthetic peptides were found to be temporary inhibitors of furin and kexin during a prolonged incubation, with the scissile peptide bond between P1 and P1' being cleaved to different extents by the enzymes. To enhance proteolytic resistance, the P1' residue Ser was mutated to D-Ser or N-methyl-Ser. The N-methyl-Ser mutant gave rise to a Ki value of 4.70x10(-8) m for furin, and retained over 80% inhibitory activity even after a 3 h incubation with the enzyme. By contrast, the d-Ser mutant was resistant to cleavage, although its inhibitory activity against furin drastically decreased. Our findings identify a useful template for the design of potent, specific and stable peptide inhibitors of furin, shedding light on the molecular determinants that dictate the inhibition of furin and kexin.     

Intracellular maturation and transport of tumor necrosis factor alpha converting enzyme

The tumor necrosis factor alpha converting enzyme (TACE) activity is required for the shedding of a variety of biologically active membrane bound precursors. The activation of TACE necessitates the proteolytic cleavage of its prodomain, a process that was suggested to be catalyzed by the proprotein convertase furin. However, the involvement of furin in this activation process has never been experimentally demonstrated. We have shown that the furinlike cleavage site (R-V-K-R(214)) localized between the prodomain and the metalloprotease domain of TACE is the sole site that can be in vitro cleaved by furin. In Cos7 cells, the release of TACE-processed substrates was reduced by the overexpression of the furin-specific proprotein convertase inhibitor Portland alpha1-antitrypsin inhibitor, but the release of TACE-processed substrates was increased by overexpression of furin in LoVo cells (deficient in furin activity) in which a mature form of TACE was identified. The immature form of TACE was detected at the surface of LoVo cells and at the surface of Cos7 and HT29 cells upon proprotein convertase inhibition. These results suggest that furin is the major proprotein convertase involved in the maturation/activation of TACE which is not a prerequisite for its cell-surface expression.     

Purification and characterization of furin, a Kex2-like processing endoprotease, produced in Chinese hamster ovary cells.

Furin, a mammalian homolog of the yeast Kex2 protease, is associated with Golgi membranes and is involved in cleavage of precursor proteins at sites marked by the Arg-X-Lys/Arg-Arg (RXK/RR) motif. We have recently shown that a furin mutant lacking the transmembrane domain can be secreted from cDNA-transfected cells with proteolytic activity for the fluorogenic peptide t-butoxycarbonyl-Arg-Val-Arg-Arg-4-methylcoumarin-7- amide. In this study, we purified and characterized the recombinant furin from the conditioned medium of these cells. Furin was purified as a mixture of 83- and 81-kDa forms and a 96-kDa form. The differences in molecular mass were not due to differences in molecular mass were not due to differences in glycosylation. Moreover, all forms had the same NH2-terminal sequence beginning at the residue after the Arg-Ala-Lys-Arg sequence. These data suggest that the three different forms may be produced by differential COOH-terminal processing of a furin molecule and that mature furin may be autocatalytically produced. Both enzyme preparations showed a pH optimum at 7.0, required Ca2+ for the activity, and showed essentially the same inhibitor profile. These properties resembled those of the Kex2 protease. Both preparations efficiently cleaved fluorogenic peptides with an RXK/RR sequence and moderately cleaved a peptide with an RXXR sequence, but did not cleave dibasic peptides. The sequence requirements determined in vitro were compatible with those determined by expression studies in cultured cells. These data unequivocally demonstrate that furin is an endogenous cellular protease responsible for cleavage of precursor proteins mainly at RXK/RR sites.