Regulatory Roles of the P Domain of the Subtilisin-like Prohormone Convertases
1998; Elsevier BV; Volume: 273; Issue: 18 Linguagem: Inglês
10.1074/jbc.273.18.11107
ISSN1083-351X
AutoresAn Zhou, Seán Martin, Gregory M. Lipkind, J LaMendola, Donald F. Steiner,
Tópico(s)Enzyme Structure and Function
ResumoA unique feature of the eukaryotic subtilisin-like proprotein convertases (SPCs) is the presence of an additional highly conserved sequence of approximately 150 residues (P domain) located immediately downstream of the catalytic domain. To study the function of this region, which is required for the production of enzymatically active convertases, we have expressed and characterized various P domain-related mutants and chimeras in HEK293 cells and α-TC1–6 cells. In a series of C-terminal truncations of PC3 (also known as PC1 or SPC3), PC3-Thr594 was identified as the shortest active form, thereby defining the functional C-terminal boundary of the P domain. Substitutions at Thr594 and nearby sites indicated that residues 592–594 are crucial for activity. Chimeric SPC proteins with interchanged P domains demonstrated dramatic changes in several properties. Compared with truncated wild-type PC3 (PC3-Asp616), both PC3/PC2Pd and PC3/FurPd had elevated activity on several synthetic substrates as well as reduced calcium ion dependence, whereas Fur/PC2Pd was only slightly decreased in activity as compared with truncated furin (Fur-Glu583). Of the three active SPC chimeras tested, all had more alkaline pH optima. When PC3/PC2Pd was expressed in α-TC1–6 cells, it accelerated the processing of proglucagon into glicentin and major proglucagon fragment and cleaved major proglucagon fragment to release GLP-1 and tGLP-1, similar to wild-type PC3. Thus, P domain exchanges generated fully active chimeric proteases in several instances but not in all (e.g. PC2/PC3Pd was inactive). The observed property changes indicate a role for the P domain in regulating the stability, calcium dependence, and pH dependence of the convertases. A unique feature of the eukaryotic subtilisin-like proprotein convertases (SPCs) is the presence of an additional highly conserved sequence of approximately 150 residues (P domain) located immediately downstream of the catalytic domain. To study the function of this region, which is required for the production of enzymatically active convertases, we have expressed and characterized various P domain-related mutants and chimeras in HEK293 cells and α-TC1–6 cells. In a series of C-terminal truncations of PC3 (also known as PC1 or SPC3), PC3-Thr594 was identified as the shortest active form, thereby defining the functional C-terminal boundary of the P domain. Substitutions at Thr594 and nearby sites indicated that residues 592–594 are crucial for activity. Chimeric SPC proteins with interchanged P domains demonstrated dramatic changes in several properties. Compared with truncated wild-type PC3 (PC3-Asp616), both PC3/PC2Pd and PC3/FurPd had elevated activity on several synthetic substrates as well as reduced calcium ion dependence, whereas Fur/PC2Pd was only slightly decreased in activity as compared with truncated furin (Fur-Glu583). Of the three active SPC chimeras tested, all had more alkaline pH optima. When PC3/PC2Pd was expressed in α-TC1–6 cells, it accelerated the processing of proglucagon into glicentin and major proglucagon fragment and cleaved major proglucagon fragment to release GLP-1 and tGLP-1, similar to wild-type PC3. Thus, P domain exchanges generated fully active chimeric proteases in several instances but not in all (e.g. PC2/PC3Pd was inactive). The observed property changes indicate a role for the P domain in regulating the stability, calcium dependence, and pH dependence of the convertases. Recently, a new family of serine proteases that process a wide variety of proprotein substrates has been identified in eukaryotic cells. Based on the similarity of their catalytic domain to the subtilisins, these proteases have been named subtilisin-like proprotein convertases (SPCs) 1The abbreviations used are: SPC, subtilisin-like prohormone convertase; peptide-MCA, peptide-methylcoumarin amide; PAGE, polyacrylamide gel electrophoresis; Bis-Tris, 2-[bis(2-hydroxyethyl) amino]-2-(hydroxymethyl)-propane-1,3-diol; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; MPGF, major proglucagon fragment; GLP-I, glucagon-like peptide I. t, truncated; pGlu, pyroglutamic acid. 1The abbreviations used are: SPC, subtilisin-like prohormone convertase; peptide-MCA, peptide-methylcoumarin amide; PAGE, polyacrylamide gel electrophoresis; Bis-Tris, 2-[bis(2-hydroxyethyl) amino]-2-(hydroxymethyl)-propane-1,3-diol; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; MPGF, major proglucagon fragment; GLP-I, glucagon-like peptide I. t, truncated; pGlu, pyroglutamic acid. (Refs.1Rouillé Y. Duguay S.J. Lund K. Furuta M. Gong Q. Lipkind G. Oliva Jr., A.A. Chan S.J. Steiner D.F. Front. Neuroendocrinol. 1995; 16: 322-361Crossref PubMed Scopus (313) Google Scholar, 2Smeekens S.P. Bio/Technology. 1993; 11: 182-186Crossref PubMed Scopus (108) Google Scholar, 3Seidah N.G. Chrétien M. Day R. Biochimie (Paris). 1994; 76: 197-209Crossref PubMed Scopus (379) Google Scholar, 4Smeekens S.P. Chan S.J. Steiner D.F. Prog. Brain Res. 1992; 92: 235-246Crossref PubMed Scopus (19) Google Scholar; for terminology see Ref. 5Chan S.J. Oliva Jr., A.A. LaMendola J. Grens A. Bode H. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6678-6682Crossref PubMed Scopus (57) Google Scholar). To date, in addition to kexin, a yeast homologue (6Julius D. Brake A. Blair L. Kunisawa R. Thorner J. Cell. 1984; 37: 1075-1089Abstract Full Text PDF PubMed Scopus (487) Google Scholar, 7Mizuno K. Nakamura T. Ohshima T. Tanaka S. Matsuo H. Biochem. Biophys. Res. Commun. 1988; 156: 246-254Crossref PubMed Scopus (178) Google Scholar) and a number of SPCs found in lower species, seven SPCs have been identified in mammals as follows: furin 2For simplicity and consistence, we refer to SPC1 as furin, SPC2 as PC2, and SPC3 as PC3 (also known as PC1) in this article. 2For simplicity and consistence, we refer to SPC1 as furin, SPC2 as PC2, and SPC3 as PC3 (also known as PC1) in this article. (PACE or SPC1), PC3 (SPC3 or PC1), PC2, PC4, PC5/PC6, PC7/PC8 (or LPC), and PACE4 (1Rouillé Y. Duguay S.J. Lund K. Furuta M. Gong Q. Lipkind G. Oliva Jr., A.A. Chan S.J. Steiner D.F. Front. Neuroendocrinol. 1995; 16: 322-361Crossref PubMed Scopus (313) Google Scholar, 8Tsuji A. Hine C. Mori K. Tamai Y. Higashine K. Nagamune H. Matsuda Y. Biochem. Biophys. Res. Commun. 1994; 202: 1452-1459Crossref PubMed Scopus (27) Google Scholar,9Bruzzaniti A. Goodge K. Jay P. Taviaux S.A. Lam M.H.C. Berta P. Martin T.J. Moseley J.M. Gillespie M.T. Biochem. J. 1996; 314: 727-731Crossref PubMed Scopus (88) Google Scholar). Each convertase has a distinct but overlapping substrate specificity and a distinctive tissue distribution, subcellular location, and maturation process, consistent with its unique role in some aspect of proprotein processing. Well characterized examples regarding these aspects are furin (expressed ubiquitously in almost all tissues), PC3, and PC2 (both restrictedly distributed in neuroendocrine tissues). We have only a limited understanding of the structural determinants which differentiate the various SPCs from each other in their function and properties. Their basic domain structure includes (Fig. 1) a signal peptide, a partially conserved propeptide, a highly conserved catalytic domain (40–50% identity among the SPCs and 25–30% to the subtilisins) followed by a relatively well conserved region called the P, homoB, or "middle" domain. Studies in recent years have clarified the role of propeptide cleavage in furin activation and the function of the propeptide as an intramolecular inhibitor which prevents early activation (10Anderson E.D. VanSlyke J.K. Thulin C.D. Jean F. Thomas G. EMBO J. 1997; 16: 1508-1518Crossref PubMed Scopus (198) Google Scholar). After the P domain, various C-terminal extensions occur; these extensions seem to contain mainly routing/trafficking determinants. For instance, furin is primarily located in the trans-Golgi network, anchored by its transmembrane domain, and mutations in its cytoplasmic domain result in dramatic changes in its subcellular location (11Molloy S.S. Thomas L. VanSlyke J.K. Stenberg P.E. Thomas G. EMBO J. 1994; 13: 18-33Crossref PubMed Scopus (419) Google Scholar, 12Dittié A.S. Thomas L. Thomas G. Tooze S.A. EMBO J. 1997; 16: 4859-4870Crossref PubMed Scopus (149) Google Scholar). Attachment of the P domain and C-terminal region of PC2 to the catalytic domain of furin resulted in a chimera which behaved similarly to PC2 in that it was now targeted to regulated pathway vesicles (13Creemers J.W.M. Usac E.F. Bright N.A. Van de Loo J.-W. Jansen E. Van de Ven W.J.M. Hutton J.C. J. Biol. Chem. 1996; 271: 25284-25291Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), whereas a truncated furin without a transmembrane domain was secreted via a non-regulated (constitutive or basal) secretory pathway. Deletion of residues from the C terminus of PC3 gave a mutant (terminating at Asp616) that was secreted in increased amounts via the constitutive pathway, whereas lesser amounts were routed into regulated secretory granules in AtT-20 cells (14Zhou A. Paquet L. Mains R.E. J. Biol. Chem. 1995; 270: 21509-21516Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The C-terminal region after Asp616 of PC3 may also function as an inhibitor (15Jutras I. Seidah N.G. Reudelhuber T.L. Brechler V. J. Biol. Chem. 1997; 272: 15184-15188Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Little is known about the function of the P domain. Due to functional differences between the subtilisins (degradative) and kexin (proteolytic processing), the conserved region after the catalytic domain of kexin was named the "P domain" when it became evident that it was required for processing activity (16Fuller R.S. Brenner C. Gluschankof P. Wilcox C.A. Jörnvall H. Höög J.-O. Gustavsson A.-M. Methods in Protein Sequence Analysis. Birkhäuser Verlag, Basel1991: 205-230Crossref Google Scholar). Although there are a few examples of bacterial subtilisins with extensions after the catalytic domain, a search of the protein data bank does not reveal any significant sequence similarity of the P domain with other known proteins. For both kexin and furin, a partial C-terminal deletion of the P domain, even of just a few amino acids, abolishes enzymatic activity completely (17Gluschankof P. Fuller R.S. EMBO J. 1994; 13: 2280-2288Crossref PubMed Scopus (86) Google Scholar, 18Creemers J.W.M. Siezen R.J. Roebroek A.J.M. Ayoubi T.A.Y. Huylebroeck D. Van de Ven W.J.M. J. Biol. Chem. 1993; 268: 21826-21834Abstract Full Text PDF PubMed Google Scholar). Lovo cells (a human colon carcinoma cell line) express furin mRNA yet lack furin activity (19Stadler K. Allison S.L. Schalich J. Heinz F.X. J. Virol. 1997; 71: 8475-8481Crossref PubMed Google Scholar). Genetic analysis demonstrated that the lack of activity was due to two mutations within the P domain coding region of the furin gene, one resulting in a frameshift (20Takahashi S. Kasai K. Hatsuzawa K. Kitamura N. Misumi Y. Ikehara Y. Murakami K. Nakayama K. Biochem. Biophys. Res. Commun. 1993; 195: 1019-1026Crossref PubMed Scopus (115) Google Scholar) and the other in a replacement of a conserved tryptophan with arginine (21Takahashi S. Nakagawa T. Kasai K. Banno T. Duguay S.J. Van de Ven W.J.M. Murakami K. Nakayama K. J. Biol. Chem. 1995; 270: 26565-26569Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Recently, a defect in the PC3 gene has been found in a human patient. In this case, a glycine within the P domain region is replaced by an arginine (22Jackson R.S. Creemers J.W.M. Ohagi S. Raffin-Sanson M.-L. Sanders L. Montague C.T. Hutton J.C. O'Rahilly S. Nat. Genet. 1997; 16: 303-306Crossref PubMed Scopus (908) Google Scholar). There has been no reported systematic study to elucidate the functional role(s) of the P domain and examine its structural relation to the catalytic domain. In the present study, using PC3 as the primary model convertase, we have investigated the properties of various P domain-related mutants and chimeras expressed in endocrine and/or non-endocrine mammalian cell lines. Our findings provide evidence that in addition to stabilizing the catalytic domain, the P domain participates in the regulation of the pH and calcium dependence, as well as the substrate specificity of these enzymes. cDNA templates for rat PC3 (PC1), PC2, and PC3-Asp616 (PC1ΔC) (14Zhou A. Paquet L. Mains R.E. J. Biol. Chem. 1995; 270: 21509-21516Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) were kindly provided by Dr. Richard Mains at the Johns Hopkins University; human furin template was from Dr. Kazuhisa Nakayama at the University of Tsukuba, Japan. pCMV6b/6c (b and c: identical except in the orientation of the polylinker region) vectors were from Dr. Graeme Bell at the University of Chicago. Unless stated otherwise, all mutants were generated by creating mutation-containing fragments by polymerase chain reaction and subcloning the polymerase chain reaction fragments into expression vectors by restriction digestion and ligation. All polymerase chain reaction-generated fragments were verified by sequencing. The orientation of subcloned fragments was verified by restriction digestion. Fig. 1 illustrates a schematic summary of the mutants used in this study. Three truncation mutants, PC3-His592, PC3-Gly593, and PC3-Thr594, terminating at amino acid His592, Gly593, and Thr594, were created by adding a stop code and a restriction site immediately after the desired amino acid, respectively. In the substitution study, Thr594 in mutant PC3-Thr594 was replaced with serine, aspartic acid, and asparagine, respectively (mutants PC3-T594S/T594D/T594N). Mutant PC3-H592T was generated by replacing His592 in PC3-His592 with threonine. Three chimeric proteins, PC3/PC2Pd, PC2/PC3Pd, and PC3/FurPd, were generated by using the "gene splicing by overlap extension" (SOE) technique (23Horton R.M. Cai Z. Ho S.N. Pease L.R. Bio/Technology. 1990; 8: 528-535Google Scholar). PC3/PC2Pd was created by fusing amino acids 1–453 of PC3 with amino acids 456–638 of PC2. PC2/PC3Pd was a fusion protein with amino acids 1–455 of PC2 and amino acids 454–616 of PC3. Mutant PC3/FurPd consists of amino acids 1–453 of PC3 and amino acids 440–583 of furin. Mutant Fur/PC2Pd (18Creemers J.W.M. Siezen R.J. Roebroek A.J.M. Ayoubi T.A.Y. Huylebroeck D. Van de Ven W.J.M. J. Biol. Chem. 1993; 268: 21826-21834Abstract Full Text PDF PubMed Google Scholar) was a kind gift from Dr. John Creemers (Katholieke Universiteit, Leuven, Belgium). A truncated PC3 (Ref. 14Zhou A. Paquet L. Mains R.E. J. Biol. Chem. 1995; 270: 21509-21516Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar; PC1ΔC, designated PC3-Asp616 in this study) and a truncated furin (Ref. 18Creemers J.W.M. Siezen R.J. Roebroek A.J.M. Ayoubi T.A.Y. Huylebroeck D. Van de Ven W.J.M. J. Biol. Chem. 1993; 268: 21826-21834Abstract Full Text PDF PubMed Google Scholar; furin Δ 476-end, designated Fur-Glu583 in the present study) were used as wild-type controls in this study. All mutants and wild-type control SPCs were cloned into either the pCMV6b or pCMV6c vector for expression in mammalian cells. HEK293 cells (human embryonic kidney tumor cells) and α-TC1–6 cells (mouse pancreatic alpha cells) were routinely maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Life Technologies, Inc.). SPC mutant vectors were transiently transfected into HEK293 cells using the calcium phosphate method (Profection kit, Promega). When stable transfection was desired, as indicated under "Results," cells were transfected using the Lipofectin method. Plasmid pSV2neo was co-transfected with the SPC mutant-carrying vectors to provide drug resistance. Transfected cells were then cultured in G418-containing medium (0.5 mg/ml); G418-resistant colonies were screened for the expression of interested SPC protein by biosynthetic labeling/immunoprecipitation, Western blotting, or immunofluorescence staining. Processing of transfected SPC protein was characterized by biosynthetic labeling. For transiently transfected cells, the labeling was performed 48 h after transfection. Briefly, cells were first incubated in methionine-deficient media for 30 min and then pulse-labeled with [35S]methionine (1 mCi/ml, 1000 Ci/mmol, Amersham Pharmacia Biotech) with or without a subsequent chase incubation in nonradioactive complete media. Upon termination of incubation, media were collected, and cellular proteins were extracted with a TES buffer containing 20 mm TES, pH 7.4, 10 mm mannitol, and 1% Triton X-100 (24Zhou A. Mains R.E. J. Biol. Chem. 1994; 269: 17440-17447Abstract Full Text PDF PubMed Google Scholar). For immunoprecipitation, samples were diluted with an immunoprecipitation buffer (50 mm Tris, pH 8.0, 250 mm NaCl, 0.5% Nonidet P-40, 0.02% NaN3) (25Melnick J. Aviel S. Argon Y. J. Biol. Chem. 1992; 267: 21303-21306Abstract Full Text PDF PubMed Google Scholar). Appropriate antibodies (as specified under "Results") and protein A (for polyclonal antibodies) or protein G (for monoclonal antibodies) (Pierce) were added. Protease inhibitors (26Milgram S.L. Mains R.E. J. Cell Sci. 1994; 107: 737-745Crossref PubMed Google Scholar) were present through the sample collection and immunoprecipitation procedures. Immunoprecipitated SPC proteins were fractionated on SDS-PAGE slab gels (7.5% or 10%) and detected by fluorography. HEK293 cells expressing various SPCs were incubated in complete serum-free Dulbecco's modified Eagle's medium for 15–20 h. Collected medium was concentrated 20–30-fold using a 30K Ultrafree concentrating unit (Millipore Corp.). Enzymatic activity assays followed the protocol described by Vindrola and Lindberg (27Vindrola O. Lindberg I. Neuropeptides. 1993; 25: 151-160Crossref PubMed Scopus (23) Google Scholar) using pGlu-Arg-Thr-Lys-Arg-MCA as the substrate. In the substrate specificity assay, Boc-Arg-Val-Arg-Arg-MCA, Boc-Leu-Ser-Thr-Arg-MCA and Boc-Ala-Gly-Pro-Arg-MCA (where Boc is t-butoxycarbonyl) were the test substrates used (Peninsula Laboratories). In experiments aimed at comparing the activity levels of various SPC chimeras, cells were cultured in duplicate plates with equal cell numbers. One plate was used to collect medium and analyze its activity. Cells in the second plate were pulse-labeled with [35S]methionine for 1 h and then chase incubated in non-radioactive complete serum-free medium for 4–6 h. Media from the second plates of various cell lines were collected, immunoprecipitated with appropriate SPC antibodies, and subjected to SDS-PAGE slab gel analysis. Upon loading the SDS-PAGE gel, each sample was mixed with non-radioactive rainbow molecular weight markers. Fractionated samples were electrically blotted onto Immobolin-P membrane (Millipore); blot areas corresponding to the appropriate molecular weight range of the processed SPCs were excised, treated with a stripping buffer (62.5 mm Tris, 2% SDS, 100 mm β-mercaptoethanol, pH 6.7) at 50 °C for 30 min, followed by scintillation counting. Preliminary tests established that loaded radioactivity could be quantitatively recovered by this procedure. Based on the radioactivity recovered from each immunoprecipitation/SDS-PAGE, after correction for the number of methionine residues in each SPC protein, the amount of medium collected from the duplicate plate used for assay of activity was adjusted so that equimolar amounts of SPC were used in each activity assay. Wild-type or transfected α-TC1–6 cells were labeled with either [35S]methionine, as described above, or [3H]leucine (0.4 mCi/ml; Amersham Pharmacia Biotech). Cellular protein extraction and immunoprecipitation were performed as described previously (28Rouillé Y. Westermark G. Martin S.K. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3242-3246Crossref PubMed Scopus (178) Google Scholar). After immunoprecipitation with the appropriate antibodies (see "Results"), [35S]methionine-labeled samples were fractionated on a gradient SDS-PAGE slab gel; [3H]leucine-labeled samples were analyzed by SDS-PAGE tube gels. Tube gels were sliced and eluted (29Zhou A. Bloomquist B.T. Mains R.E. J. Biol. Chem. 1993; 268: 1763-1769Abstract Full Text PDF PubMed Google Scholar), followed by scintillation counting. Non-radioactive rainbow molecular weight markers were added with samples as internal standards. Among the known mammalian SPCs, PC3-Asp616, which migrated as a 67-kDa species in the present study (Fig. 2), is the shortest active form. It is normally produced in neuroendocrine cells after the autocatalytic removal of the propeptide in the endoplasmic reticulum and the subsequent deletion of a C-terminal fragment (amino acids 617–736) in the secretory granules (14Zhou A. Paquet L. Mains R.E. J. Biol. Chem. 1995; 270: 21509-21516Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 30Zhou Y. Rovere C. Kitabgi P. Lindberg I. J. Biol. Chem. 1995; 270: 24702-24706Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Non-endocrine cells usually do not endogenously express PC3. Its relative simplicity, selective distribution, and our greater understanding of its maturation process and function make this truncated form of PC3 an ideal model convertase for studies on the P domain. Of the mammalian P domains, the last two conserved amino acids in the C-terminal region, presumably marking their junction with the C-terminal extensions, are Gly593 and Thr594(numbers are for rat PC3). PC3-Asp616 contains an additional 22 amino acids beyond this conserved region. To define more precisely the shortest sequence needed for activity, three truncation mutants ending at or near these conserved amino acids were expressed in HEK293 cells. Their propeptide cleavage activity and secretion were examined by a pulse-chase metabolic labeling paradigm (Fig. 2). PC3-Asp616 undergoes very efficient autocatalytic cleavage to remove its propeptide. During a 30-min pulse-labeling period, the majority of 75-kDa pro-PC3-Asp616 was converted to 67-kDa mature PC3, and most of the latter was secreted into medium during the subsequent 3-h chase incubation. For truncation mutant PC3-Thr594 (Fig. 2, right), the dominant form during the 30-min pulse was a molecule slightly smaller than 75 kDa. Later, it was processed into a 65-kDa protein, an expected size for this truncated PC3 after removal of its propeptide. The conversion rate of pro-PC3-Thr594 to PC3-Thr594 was not as efficient as that of pro-PC3-Asp616 to PC3-Asp616, but after 3 h of chase incubation, a significant amount of the processed PC3-Thr594 was secreted into the medium. In contrast, neither PC3-Gly593 (Fig. 2,middle) nor PC3-His592 (data not shown) was processed or secreted. To determine whether the lack of propeptide cleavage in PC3-Gly593 and PC3-His592 was due to the absence of threonine at the C terminus of the P domain, Thr594 in PC3-Thr594 was replaced with serine, aspartic acid, or asparagine, respectively. Fig. 3 shows a biosynthetic labeling experiment on HEK293 cells expressing PC3-T594S, T594D, and T594N. After a 30-min pulse and a 3-h chase incubation, a small amount of mature 65-kDa protein was seen in the media of cells expressing PC3-T594S and PC3-T594N, whereas it was not visible with mutant PC3-T594D. For mutant PC3-H592T, in which His592 in the PC3-His592 mutant was replaced with threonine, there was no detectable propeptide cleavage activity. These results thus demonstrated that the shortest form of PC3 capable of cleaving its propeptide is PC3-Thr594. The few conserved amino acids at the C terminus of the P domain region (His592, Gly593, and Thr594) also seem to play a crucial role in sustaining the autocatalytic activity of PC3. This also confirmed that the P domain of PC3 has a C-terminal well defined boundary. The relatively high level of conservation of the P domains among various SPCs suggests that these domains must play some essential role. We therefore reasoned that interchanges of P domains might provide information on these possible function(s). Accordingly, P domain-swapped SPCs were prepared and transfected into HEK293 cells (Fig. 1). The autocatalytic activation of each chimeric form was studied by pulse-chase metabolic labeling, and its processing and secretion were compared with that of the parental proteases. PC3/PC2Pd was initially synthesized as a 79-kDa protein, which was then converted into a 69-kDa protein through an intermediate (Fig. 4, top panel). The conversion rate was slower than that of the propeptide processing of PC3-Asp616. The secretion of 69-kDa PC3/PC2Pd protein, however, was as efficient as the secretion of PC3-Asp616. The counterpart mutant, PC2/PC3Pd, did not show any propeptide cleavage activity (Fig. 4, bottom panel), even when 7B2 (a unique PC2 helper protein) (31Muller L. Zhu X. Lindberg I. J. Cell Biol. 1997; 139: 625-638Crossref PubMed Scopus (87) Google Scholar, 32Benjannet S. Savaria D. Chrétien M. Seidah N.G. J. Neurochem. 1995; 64: 2303-2311Crossref PubMed Scopus (90) Google Scholar, 33Lamango N.S. Zhu X. Lindberg I. Arch. Biochem. Biophys. 1996; 330: 238-250Crossref PubMed Scopus (86) Google Scholar) was co-expressed and the chase incubation was extended to 8 h (data not shown). Mutant PC3/FurPd also displayed propeptide cleavage activity, although it was much less efficient than that of PC3-Asp616, and the secretion of the processed form was relatively slow (Fig. 4,middle panel). The presence of two forms in the cells after a 3-hour chase presumably represents differences in glycosylation status. During the course of this study, Creemers et al.(13Creemers J.W.M. Usac E.F. Bright N.A. Van de Loo J.-W. Jansen E. Van de Ven W.J.M. Hutton J.C. J. Biol. Chem. 1996; 271: 25284-25291Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) reported that, in their study on the sorting mechanisms of SPCs, a chimeric protein with the catalytic domain of furin and the P domain of PC2 underwent efficient propeptide cleavage and secretion via the regulated secretory pathway in AtT-20 cells. When we expressed this mutant in HEK293 cells, it also showed efficient propeptide cleavage and secretion, similar to that of Fur-Glu583 (data not shown). The successful propeptide cleavage and secretion of the chimeric SPCs suggested that they were able to fold and be transported in HEK293 cells. Next we attempted to determine whether these chimeric enzymes were active in cleaving substrates. Our preliminary experiments established that wild-type HEK293 cells in our culture system secrete negligible amounts of endoproteolytic activity toward MCA-linked synthetic substrates, as tested over a range of pH values and varying calcium ion concentrations. Collected media samples from HEK293 cells expressing various SPC mutants and wild-type SPCs were analyzed for their activities against fluorogenic substrates. PC3/PC2Pd, PC3/FurPd, and Fur/PC2Pd were all active (see below). Three major enzymatic properties that distinguish eukaryotic SPCs from bacterial subtilisins are their dependence on calcium for activation and activity, their more acidic pH optima, and their requirement of two or more basic amino acids at the substrate cleavage site. Efforts were made to address each of these properties in the mutants. We first tested the calcium dependence of the active chimeras. Between 0 and 20 mmcalcium ion concentration, PC3-Asp616 showed a gradual increase of activity. The activity level plateaued above 20 mm calcium ion concentration. Omitting calcium and adding 2 mm EGTA or 2 mm EDTA to the assay completely suppressed the activity of PC3-Asp616 (Fig. 5, top panel). Quite strikingly, PC3/PC2Pd retained 40–55% of its activity in the presence of EGTA or EDTA, and its activity increased only moderately over the range from 0 to 40 mm calcium ion concentration. PC3/FurPd (Fig. 5, top panel), on the other hand, had little activity in the presence of EGTA or EDTA. It gained most of its activity in the lower range of calcium concentration (0–5 mm). The activity of Fur/PC2Pd responded to the changes in calcium ion concentration in a pattern similar to that of Fur-Glu583(Fig. 5, bottom panel), i.e. no activity in the presence of EGTA or EDTA and gaining most of the activity in the low range of calcium ion concentrations. Full-size PC3 and C-terminally processed PC3 have different acidic pH optima (34Zhou Y. Lindberg I. J. Biol. Chem. 1994; 269: 18408-18413Abstract Full Text PDF PubMed Google Scholar, 35Coates L.C. Birch N.P. J. Neurochem. 1997; 68: 828-836Crossref PubMed Scopus (14) Google Scholar). Similar to other published results, PC3-Asp616 secreted from HEK293 cells showed maximal activity at pH 6.0 within a narrow range (Fig. 6, top panel). Fur-Glu583 had a broader optimum pH, ranging between pH 7 and pH 8 (Fig. 6, bottom panel). The optimum pH for PC3/PC2Pd was shifted to between pH 7.0 and pH 8.0 (Fig. 6, top panel). The optimum pH for PC3/FurPd was pH 7.0 (Fig. 6, top panel), in between that of Fur-Glu583 and PC3-Asp616. Fur/PC2Pd demonstrated a more alkaline and narrower optimum pH (pH 8.0) than was observed for Fur-Glu583 (Fig. 6, bottom panel). Taken together, all three chimeric SPC proteins had more alkaline pH optima than their parental wild types. The activity levels of various SPC chimeras with fluorogenic substrates were compared at their optimum pH and with 10 mm calcium (Table I, top). The specific activities of PC3/PC2Pd and PC3/FurPd were 3–4-fold higher than the activity of PC3-Asp616, respectively, whereas the activity of Fur/PC2Pd was slightly lower than the activity of Fur-Glu583. PC3-Thr594 had a level of activity similar to that of PC3-Asp616 (data not shown); PC3-T594S and PC3-T594N, although secreted in minor amounts, did not show any detectable enzymatic activity (data not
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