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• W2004671440 abstract "Several recently discovered members of the carboxypeptidase E (CPE) gene family lack critical active site residues that are conserved in other family members. For example, three CPE-like proteins contain a Tyr in place of Glu300 (equivalent to Glu270 of carboxypeptidase A and B). To investigate the importance of this position, Glu300 of rat CPE was converted into Gln, Lys, or Tyr, and the proteins expressed in Sf9 cells using the baculovirus system. All three mutants were secreted from the cells, but the media showed no enzyme activity above background levels. Wild-type CPE and the Gln300 point mutant bound to a p-aminobenzoyl-Arg-Sepharose affinity resin, and this binding was competed by an active site-directed inhibitor, guanidinoethylmercaptosuccinic acid. The affinity purified mutant CPE protein showed no detectable enzyme activity (<0.004% of wild-type CPE) toward dansyl-Phe-Ala-Arg. Expression of the Gln300 and Lys300 mutant CPE proteins in the NIT3 mouse pancreatic beta-cell line showed that these mutants are routed into secretory vesicles and secreted via the regulated pathway. Taken together, these results indicate that Glu300 of CPE is essential for enzyme activity, but not required for substrate binding or for routing into the regulated secretory pathway. Several recently discovered members of the carboxypeptidase E (CPE) gene family lack critical active site residues that are conserved in other family members. For example, three CPE-like proteins contain a Tyr in place of Glu300 (equivalent to Glu270 of carboxypeptidase A and B). To investigate the importance of this position, Glu300 of rat CPE was converted into Gln, Lys, or Tyr, and the proteins expressed in Sf9 cells using the baculovirus system. All three mutants were secreted from the cells, but the media showed no enzyme activity above background levels. Wild-type CPE and the Gln300 point mutant bound to a p-aminobenzoyl-Arg-Sepharose affinity resin, and this binding was competed by an active site-directed inhibitor, guanidinoethylmercaptosuccinic acid. The affinity purified mutant CPE protein showed no detectable enzyme activity (<0.004% of wild-type CPE) toward dansyl-Phe-Ala-Arg. Expression of the Gln300 and Lys300 mutant CPE proteins in the NIT3 mouse pancreatic beta-cell line showed that these mutants are routed into secretory vesicles and secreted via the regulated pathway. Taken together, these results indicate that Glu300 of CPE is essential for enzyme activity, but not required for substrate binding or for routing into the regulated secretory pathway. carboxypeptidase A carboxypeptidase B carboxypeptidase D hemagglutinin guanidinoethylmercaptosuccinic acid phosphate-buffered saline Metallocarboxypeptidases perform many physiological functions. Pancreatic carboxypeptidases A and B (CPA1 and CPB) are primarily involved with the digestion of food, whereas CPE and related family members perform more selective cleavages and are involved in the biosynthesis of peptides and proteins (1Fricker L.D. Annu. Rev. Physiol. 1988; 50: 309-321Crossref PubMed Scopus (307) Google Scholar). Altogether, there are 13 members of this gene family, which is subdivided into two groups based on homology and size. Each group has 30–70% amino acid sequence identity with other members of the same group, but only 15–25% amino acid sequence identity with members of the other group. One group includes pancreatic CPA1, CPA2, CPB, mast cell CPA, and carboxypeptidase U (also known as plasma CPB) (2Bradshaw R.A. Ericsson L.H. Walsh K.A. Neurath H. Proc. Natl. Acad. Sci. U. S. A. 1969; 63: 1389-1394Crossref PubMed Scopus (142) Google Scholar, 3Gardell S.J. Craik C.S. Clauser E. Goldsmith E.J. Stewart C.B. Graf M. Rutter W.J. J. Biol. Chem. 1988; 263: 17828-17836Abstract Full Text PDF PubMed Google Scholar, 4Titani K. Ericsson L.H. Walsh K.A. Neurath H. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 1666-1670Crossref PubMed Scopus (66) Google Scholar, 5Reynolds D.S. Stevens R.L. Gurley D.S. Lane W.S. Austen K.F. Serafin W.E. J. Biol. Chem. 1989; 264: 20094-20099Abstract Full Text PDF PubMed Google Scholar, 6Eaton D.L. Malloy B.E. Tsai S.P. Henzel W. Drayna D. J. Biol. Chem. 1991; 266: 21833-21838Abstract Full Text PDF PubMed Google Scholar). These family members are all enzymatically active, and are 30–40-kDa proteins. The other group includes CPE, carboxypeptidase M (CPM), carboxypeptidase N (CPN), carboxypeptidase D (CPD), carboxypeptidase Z (CPZ), and three proteins designated CPX1, CPX2, and AEBP1 (7Fricker L.D. Evans C.J. Esch F.S. Herbert E. Nature. 1986; 323: 461-464Crossref PubMed Scopus (141) Google Scholar, 8Tan F. Chan S.J. Steiner D.F. Schilling J.W. Skidgel R.A. J. Biol. Chem. 1989; 264: 13165-13170Abstract Full Text PDF PubMed Google Scholar, 9Gebhard W. Schube M. Eulitz M. Eur. J. Biochem. 1989; 178: 603-607Crossref PubMed Scopus (41) Google Scholar, 10Kuroki K. Eng F. Ishikawa T. Turck C. Harada F. Ganem D. J. Biol. Chem. 1995; 270: 15022-15028Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 11Song L. Fricker L.D. J. Biol. Chem. 1995; 270: 25007-25013Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 12Xin X. Varlamov O. Day R. Dong W. Bridgett M.M. Leiter E.H. Fricker L.D. DNA Cell Biol. 1997; 16: 897-909Crossref PubMed Scopus (75) Google Scholar, 13Song L. Fricker L.D. J. Biol. Chem. 1997; 272: 10543-10550Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 14Xin X. Day R. Dong W. Lei Y. Fricker L.D. DNA Cell Biol. 1998; 17: 897-909Crossref PubMed Scopus (47) Google Scholar, 15Lei Y. Xin X. Morgan D. Pintar J.E. Fricker L.D. DNA Cell Biol. 1999; 18: 175-185Crossref PubMed Scopus (45) Google Scholar, 16He G.P. Muise A. Li A.W. Ro H.S. Nature. 1995; 378: 92-96Crossref PubMed Scopus (135) Google Scholar, 17Layne M.D. Endege W.O. Jain M.K. Yet S.-F. Hsieh C.-M. Chin M.T. Perrella M.A. Blanar M.A. Haber E. Lee M.-E. J. Biol. Chem. 1998; 273: 15654-15660Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Except for CPD, which is 180 kDa and contains 3 distinct carboxypeptidase-like domains, the members of this family contain a single carboxypeptidase domain of approximately 400 amino acids. Only CPE, CPM, CPN, CPZ, and the first two domains of CPD have been demonstrated to have enzymatic activity; the third domain of CPD and the other proteins in this group do not appear to cleave the standard carboxypeptidase substrates (13Song L. Fricker L.D. J. Biol. Chem. 1997; 272: 10543-10550Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 14Xin X. Day R. Dong W. Lei Y. Fricker L.D. DNA Cell Biol. 1998; 17: 897-909Crossref PubMed Scopus (47) Google Scholar, 15Lei Y. Xin X. Morgan D. Pintar J.E. Fricker L.D. DNA Cell Biol. 1999; 18: 175-185Crossref PubMed Scopus (45) Google Scholar,18Eng F.J. Novikova E.G. Kuroki K. Ganem D. Fricker L.D. J. Biol. Chem. 1998; 273: 8382-8388Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar).The observation that CPX2, AEBP1, and the third domain of CPD are not active toward standard substrates is consistent with a comparison of active site residues of the various members of the family. CPX2, AEBP1, and the third domain of CPD lack a Glu in the position equivalent to Glu270 of CPA and CPB. Instead, these novel members of the gene family contain Tyr in this position (Fig.1). Glu270 in CPA/B is a critical catalytic residue that is thought to contribute a proton to the leaving group following hydrolysis of the amide bond (19Christianson D.W. Adv. Protein Chem. 1991; 42: 281-355Crossref PubMed Google Scholar). Theoretically, it is possible that a Tyr in this position could also function as a proton donor, but the catalytic activity would be predicted to be greatly reduced. However, substrate binding may not be greatly affected by the absence of a Glu in this position. These observations led to the prediction that the novel members of the metallocarboxypeptidase gene family that lack the Glu270equivalent residue are binding proteins, rather than active enzymes.In the present study, we tested the importance of Glu300 in rat CPE. In addition to testing the affect on enzyme activity and binding to a substrate affinity resin, we also tested whether the mutant CPE could be correctly sorted into the regulated secretory pathway in a CPE-deficient pancreatic beta cell line. Recently, CPE has been proposed to be involved in the sorting of neuroendocrine peptides into the regulated secretory pathway (20Cool D.R. Normant E. Shen F. Chen H. Pannell L. Zhang Y. Loh Y.P. Cell. 1997; 88: 73-83Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). Thus, we were interested in testing whether mutations that inactivate CPE had an influence on the sorting of CPE in a neuroendocrine cell line. For this analysis, we used the NIT3 cell line (21Varlamov O. Fricker L.D. Furukawa H. Steiner D.F. Langley S.H. Leiter E.H. Endocrinology. 1997; 138: 4883-4892Crossref PubMed Scopus (60) Google Scholar), which was derived fromCpe fat /Cpe fat mice and does not contain active CPE due to a point mutation of Ser202 to Pro in the coding region of the CPE gene (22Naggert J.K. Fricker L.D. Varlamov O. Nishina P.M. Rouille Y. Steiner D.F. Carroll R.J. Paigen B.J. Leiter E.H. Nat. Genet. 1995; 10: 135-142Crossref PubMed Scopus (607) Google Scholar). This cell line is an ideal test system to examine the sorting of CPE because it contains a regulated secretory pathway without endogenous CPE (the Pro202 mutant CPE is degraded before entry into the Golgi) (21Varlamov O. Fricker L.D. Furukawa H. Steiner D.F. Langley S.H. Leiter E.H. Endocrinology. 1997; 138: 4883-4892Crossref PubMed Scopus (60) Google Scholar). Our findings that mutations of Glu300 eliminate CPE activity but not binding to a substrate affinity column supports the hypothesis that the novel family members lacking a Glu in the equivalent position are binding proteins rather than active enzymes. Furthermore, our finding that the CPE proteins with mutations of Glu300 are sorted into the regulated pathway in the NIT3 cells indicates that neither enzyme activity or substrate binding are required for this sorting.RESULTSWild type CPE has previously been expressed in high levels in Sf9 cells using the baculovirus system (24Varlamov O. Leiter E.H. Fricker L.D. J. Biol. Chem. 1996; 271: 13981-13986Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Mutation of the Glu300 into Lys, Gln, or Tyr does not affect the relative levels of CPE protein expression in the baculovirus system (Fig.2). High levels of CPE protein are detected in both cells and media for all three point mutants. However, enzymatic activity toward dansyl-Phe-Ala-Arg is detectable only for the wild-type CPE (Table I). The level of enzyme activity of the wild-type CPE is similar to that determined in previous studies (13Song L. Fricker L.D. J. Biol. Chem. 1997; 272: 10543-10550Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The three point mutants show only background activity that is comparable with controls with nonexpressing virus (Table I). Similar results were observed in three separate experiments. Because of the presence of low levels of endogenous activities in the Sf9 cells, studies on crude cells or media cannot rule out the possibility that the mutant proteins have low levels of enzyme activity. To investigate this further, purification of the proteins were attempted on a substrate affinity resin.Figure 2Western blot analysis of wild-type CPE (E-300) and various point mutants upon expression using the baculovirus system. Aliquots of cell (C) and media (M) after infection of Sf9 cells with recombinant baculovirus-expressing CPE were analyzed by Western blotting as described under “Materials and Methods.” Con, wild-type virus control. The molecular masses of prestained standards (Life Technologies, Inc.) are indicated.View Large Image Figure ViewerDownload (PPT)Table IActivities of wild type CPE (Glu 300) and point mutations of Glu 300 upon expression in Sf9 cells using the baculovirus expression systemGlu300Gln300Lys300Tyr300Control virusCell extracts36.9<0.2<0.2<0.2<0.2Media15.8<0.1<0.1<0.1<0.1Activity was measured at pH 5.5 using 0.2 mmdansyl-Phe-Ala-Arg; units are nanomoles of product formed/min/106 cells. Open table in a new tab When applied to a Sepharose column containingp-aminobenzoyl-Arg, the majority of the wild-type CPE is bound and only trace levels are detected in the flow through fraction (Fig. 3, F). Raising the pH to 8.0 causes most of the bound CPE to elute from the column (Fig. 3,E1). Similar analysis of the three point mutants showed that only the Gln300 form of CPE was able to bind to the affinity resin, and subsequently elute with high pH buffer (Fig. 3). Both the Lys300 and the Tyr300 mutants were unable to bind to the affinity resin. To test whether a small amount of protein bound to the column but could not be eluted by the high pH, the columns were also eluted with a high concentration of Arg after the high pH treatment. This treatment, which elutes other carboxypeptidases such as CPD, did not elute any detectable amount of Lys300or Tyr300 CPE (Fig. 3, E2). Thus, these two mutants do not appear able to bind to the substrate affinity resin, whereas the Gln300 mutant is able to bind.Figure 3Western blot analysis of the binding of wild-type CPE and various point mutants to a substrate affinity column. CPE expression media (S) from Sf9 cells were applied to p-aminobenzoyl-Arg-Sepharose 6B affinity columns as described under “Materials and Methods.” The flow through (F) represents the fraction of the media passing through the column unretained. The binding proteins were eluted first with pH 8.0 Tris-Cl buffer (E1), then with 25 mmarginine in the pH 8.0 buffer (E2). The entire experiment was repeated three times with comparable results. The molecular masses of prestained standards (Life Technologies, Inc.) are indicated.View Large Image Figure ViewerDownload (PPT)To ensure that the binding of the Gln300 form of CPE to thep-aminobenzoyl-Arg-Sepharose represents binding to the active site of the protein, we tested whether an active site-directed inhibitor could compete for this binding. GEMSA is a competitive inhibitor of CPE with K i around 10 nm(29Fricker L.D. Plummer T.H.J. Snyder S.H. Biochem. Biophys. Res. Commun. 1983; 111: 994-1000Crossref PubMed Scopus (61) Google Scholar). When affinity column-bound wild-type or Gln300 CPE was incubated with 0.1 mm GEMSA, nearly all of the bound protein was eluted. To quantitate this, the experiment was performed with various concentrations of GEMSA. Approximately 50% of the wild-type CPE is eluted from the p-aminobenzoyl-Arg column with 6 μm GEMSA (Fig. 4). Similar analysis of the Gln300 form of CPE shows that 2 μm GEMSA elutes 50% of the bound protein (Fig. 4). Thus, both wild-type and Gln300 CPE bind to the affinity column through their active sites, and this binding is competed by generally similar concentrations of an active site-directed inhibitor.Figure 4Competition of the binding of CPE to the substrate affinity resin with GEMSA. Wild-type CPE (E300) and a mutant (Q300) were loaded onto the affinity column, and the protein was eluted with various concentrations of GEMSA at pH 5.5 as described under “Materials and Methods.” The columns were subsequently eluted with pH 8.0 buffer, and the amount of CPE in each elute was quantitated by analysis on a polyacrylamide gel followed by silver staining and densitization of the protein bands. The level of CPE in the first eluate is shown as a percentage of the total amount of CPE bound to each column. The experiment was performed twice, and the average of the two experiments is shown. Error bars show the range of these two determinations.View Large Image Figure ViewerDownload (PPT)Following purification of the Gln300 form of CPE on the affinity columns, the activity toward the standard carboxypeptidase substrate dansyl-Phe-Ala-Arg was tested. For this analysis, the affinity column-bound protein was eluted by raising the pH to 8.0 (as done in Fig. 3) rather than with GEMSA (as done in Fig. 4) because GEMSA would interfere with the enzyme activity measurement. Wild type CPE showed a V max of 20 μmol/min/mg protein, whereas no activity could be detected for the Gln300 form of CPE. Based on the sensitivity of the assay and the amount of purified protein tested, the Gln300 CPE has less than 0.004% of the activity of wild-type CPE.To test whether CPE activity or substrate binding are required for sorting of CPE into the regulated pathway, we expressed the proteins in the NIT3 pancreatic cell line. For this analysis, the HA epitope was added to the N terminus of the CPE so that the expressed protein could be detected above the background of endogenous CPE, which is only detected in the endoplasmic reticulum of these cells due to the naturally occurring point mutation (21Varlamov O. Fricker L.D. Furukawa H. Steiner D.F. Langley S.H. Leiter E.H. Endocrinology. 1997; 138: 4883-4892Crossref PubMed Scopus (60) Google Scholar). The cells were co-transfected with a plasmid expressing rat prodynorphin so that the distribution of the CPE could be compared with that of a prohormone. Prodynorphin has been previously found to be routed into the secretory pathway of neuroendocrine cells (30Devi L. Gupta P. J. Neuroendocrinology. 1989; 1: 363-368Crossref PubMed Scopus (2) Google Scholar, 31Devi L. Gupta P. Douglass J.D. Mol. Endocrinol. 1989; 3: 1852-1860Crossref PubMed Scopus (16) Google Scholar). When expressed in NIT3 cells, the epitope tagged wild-type CPE (Glu300 showed a punctate pattern throughout the cytoplasm) (Fig. 5). Co-staining of the cells with a rabbit polyclonal antiserum to prodynorphin showed a similar pattern of distribution (Fig. 5). This pattern is consistent with localization of the CPE to secretory vesicles in this cell type. The distribution of the Lys300and Gln300 forms of CPE also showed overlap to the prodynorphin (Fig. 5), suggesting that they were also routed to the secretory vesicles.Figure 5Immunocytochemistry of NIT3 cells expressing epitope-tagged wild-type CPE (E 300 ) or various point mutations. Cells were transiently co-transfected with cDNA encoding HA-tagged CPE and prodynorphin and then stained with antibodies to HA epitope-tag (left column) and the 13 S antiserum to dynorphin B-13 (right column) as described under “Materials and Methods.” Scale bar: 10 μm.View Large Image Figure ViewerDownload (PPT)To directly examine if the expressed CPE was routed to the regulated secretory pathway, the media of the cells were examined following stimulation with 45 mm KCl. In the absence of stimulation, extremely low levels of CPE protein are detected in the media (Fig.6). Following stimulation with KCl, there is a substantial increase in the amount of CPE detected in the media (Fig. 6). Quantitation of the results from three experiments shows the secretion of the Gln300 and Lys300forms of CPE to be stimulated by KCl to a similar degree as the epitope-tagged wild-type Glu300 form of CPE. Thus, enzyme activity or substrate binding do not appear to be required for entry of CPE into the regulated secretory pathway.Figure 6Regulated secretion of wild-type CPE (E 300 ) and point mutants from NIT3 cells.Cells stably transfected with different HA-tagged CPE constructs were incubated for 40 min either in control media or with 45 mmKCl, and then the media were analyzed on a Western blot using antibodies to the HA epitope. Similar results were obtained with three separate clones for each construct.View Large Image Figure ViewerDownload (PPT)DISCUSSIONA major finding of this study is that Glu300 of CPE is essential for enzyme activity but not for substrate binding. The critical nature of this residue was predicted from previous studies on the structure and function of CPA and CPB, but due to the relatively low conservation of amino acids between CPE and the CPA/B subfamily, it was important to test this directly. In CPA and CPB, the Glu in a comparable position (Glu270 is thought to be responsible for the protonation of the leaving group following hydrolysis of the amide bond (19Christianson D.W. Adv. Protein Chem. 1991; 42: 281-355Crossref PubMed Google Scholar). In other hydrolases, such as β-lactamases, some members of the gene family contain a Glu in a position thought to function as a general base, whereas other family members contain a Tyr in a comparable position (32Philippon A. Dusart J. Joris B. Frere J.M. Cell. Mol. Life Sci. 1998; 54: 341-346Crossref PubMed Scopus (60) Google Scholar). Thus, Tyr is potentially able to function in this capacity in the novel members of the CPE gene family.In this study, replacement of the Glu300 of CPE with Tyr resulted in an inactive enzyme that did not bind to the substrate affinity column. It is possible that the bulky side chain of the Tyr caused changes in the active site that precluded substrate binding. The substitution of Gln for Glu is relatively minor, changing only the -COOH into a -CONH2. This smaller change preserved binding to the substrate affinity column, indicating that binding does not require Glu300. Even though the Tyr300 and Lys300 forms of CPE were not able to bind to the affinity resin, these forms were secreted from both the Sf9 cells, and the Lys300 CPE was secreted from NIT3 cells (Tyr300 CPE was not examined in NIT3 cells). In contrast, forms of CPE containing deletions of 33 or more C-terminal residues, or point mutation of Ser202 into Pro are not secreted from either cell type and are degraded before protein transport into the Golgi (24Varlamov O. Leiter E.H. Fricker L.D. J. Biol. Chem. 1996; 271: 13981-13986Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 33Varlamov O. Fricker L.D. J. Biol. Chem. 1996; 271: 6077-6083Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The failure of these other mutations of CPE to be secreted presumably reflects the improper folding of these proteins. Because the Tyr300 and Lys300 forms of CPE were secreted in levels comparable with wild-type CPE, these point mutants are unlikely to be completely misfolded.The regulated secretion of Gln300 and Lys300CPE in the NIT3 cells indicates that neither enzyme activity or substrate binding are required for entry into the regulated pathway. Recently, Y. P. Loh and colleagues (20Cool D.R. Normant E. Shen F. Chen H. Pannell L. Zhang Y. Loh Y.P. Cell. 1997; 88: 73-83Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 34Normant E. Loh Y.P. Endocrinology. 1998; 139: 2137-2145Crossref PubMed Scopus (73) Google Scholar) have proposed that CPE is a hormone-sorting receptor. This proposal is based on studies examining the sorting of prohormones in the fat/fat mouse, and in cell lines that have a reduced level of CPE protein due to the expression of antisense RNA. However, proinsulin sorting is not dependent on CPE, based on studies of primary cultures of pancreatic beta cells (35Irminger J.-C. Verchere C.B. Meyer K. Halban P.A. J. Biol. Chem. 1997; 272: 27532-27534Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) or the NIT3 beta cell line (21Varlamov O. Fricker L.D. Furukawa H. Steiner D.F. Langley S.H. Leiter E.H. Endocrinology. 1997; 138: 4883-4892Crossref PubMed Scopus (60) Google Scholar). The sorting of proinsulin and insulin into the regulated pathway of the beta cells proceeds in the complete absence of CPE from this pathway. Also, previous studies have found that prodynorphin is routed to the regulated secretory pathway in the NIT3 cell line. 2L. Devi, personal communication. Thus, CPE is not essential for the sorting of prohormones in pancreatic beta cells, although it is possible that CPE contributes to this process in other cell types. The mechanism by which CPE is sorted into the regulated pathway has not been elucidated. The C-terminal region of CPE appears to contain an element that contributes to the sorting into the regulated pathway; attachment of the C-terminal 50 residues to albumin causes a portion of the fusion protein to be secreted via the regulated pathway (33Varlamov O. Fricker L.D. J. Biol. Chem. 1996; 271: 6077-6083Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). This region is distinct from the membrane-binding motif also present in the C-terminal region of CPE (36Mitra A. Song L. Fricker L.D. J. Biol. Chem. 1994; 269: 19876-19881Abstract Full Text PDF PubMed Google Scholar). CPE aggregates at slightly acidic pH values, and this aggregation is enhanced by Ca2+ (37Nalamachu S.R. Song L. Fricker L.D. J. Biol. Chem. 1994; 269: 11192-11195Abstract Full Text PDF PubMed Google Scholar, 38Song L. Fricker L.D. J. Biol. Chem. 1995; 270: 7963-7967Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar); it is possible that this aggregation contributes to the routing of CPE. Models of co-aggregation of the secretory vesicle constituents have been proposed to account for the routing of the proteins into the regulated pathway (39Chanat E. Huttner W.B. J. Cell Biol. 1991; 115: 1505-1519Crossref PubMed Scopus (386) Google Scholar, 40Bauerfeind R. Huttner W.B. Curr. Opin. Cell Biol. 1993; 5: 628-635Crossref PubMed Scopus (135) Google Scholar). Because the mutants of CPE that do not bind to the substrate affinity column are correctly routed in the NIT3 cells, the mechanism of CPE sorting presumably does not involve substrate binding to the active site.The finding that Glu300 is not essential for binding of CPE to the affinity column supports the hypothesis that the new members of the CPE gene family that lack a Glu in the comparable position function as binding proteins rather than active enzymes. These three proteins (CPX2, AEBP1, and the third CP-like domain of CPD) have not been found to have enzyme activity upon expression in Sf9 cells using the baculovirus system (13Song L. Fricker L.D. J. Biol. Chem. 1997; 272: 10543-10550Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 14Xin X. Day R. Dong W. Lei Y. Fricker L.D. DNA Cell Biol. 1998; 17: 897-909Crossref PubMed Scopus (47) Google Scholar, 18Eng F.J. Novikova E.G. Kuroki K. Ganem D. Fricker L.D. J. Biol. Chem. 1998; 273: 8382-8388Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Although AEBP1 was reported to have detectable activity upon expression in bacteria (16He G.P. Muise A. Li A.W. Ro H.S. Nature. 1995; 378: 92-96Crossref PubMed Scopus (135) Google Scholar), this activity was extremely low (0.02 absorption units at 256 nm with the substrate hippuryl-Arg after 10 min with 2 μg of enzyme). Furthermore, attempts to repeat the experiment using the identical constructs, expression system, and assay were unsuccessful. 3Y. Qian, O. Varlamov, and L. D. Fricker, unpublished results. However, it remains possible that these proteins are active enzymes. The substitution of the Tyr for Glu300 of CPE was created with the hope that the protein would still bind to the affinity column and thus be able to be purified and tested with substrate in the absence of other enzymes present in the Sf9 cells and media. This was done for the Gln300 mutant, with the finding that this variant had less than 0.004% of the activity of wild-type CPE. However, as discussed above, the bulky side chain of the Tyr may have altered the active site and prevented binding to the affinity resin. Although alternative purification strategies could have been used to isolate the enzyme, the failure to bind to the substrate affinity resin suggests that the protein would not bind substrates and would therefore be unlikely to possess enzyme activity. The lack of enzyme activity in the media or cell extracts of the Sf9 cells infected with Tyr300 CPE-expressing baculovirus indicates that this mutant has less than 0.6% of the wild-type CPE activity.In summary, Glu300 of CPE is essential for enzyme activity but not substrate binding. The recent identification of three members of the CPE gene family that lack a Glu in a comparable position highlights the importance of this residue. Interestingly, several other enzyme families contain members with substitutions of critical active site residues that affect enzyme activity but not substrate binding. For example, aspartyl protease-like proteins that lack the critical Asp have been found in bovine and sheep placenta (41Roberts R.M. Xie S. Nagel R.J. Low B. Green J. Beckers J.F. Adv. Exp. Med. Biol. 1995; 362: 231-240Crossref PubMed Scopus (26) Google Scholar, 42Xie S. Green J. Bixby J.B. Szafranska B. DeMartini J.C. Hecht S. Roberts R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12809-12816Crossref PubMed Scopus (152) Google Scholar), and disintegrin-like proteins that lack the consensus site for metal binding have been found in human brain, testis, and other tissues (43Sagane K. Ohya Y. Hasegawa Y. Tanaka I. Biochem. J. 1998; 334: 93-98Crossref PubMed Scopus (73) Google Scholar,44van Huijsduijnen R.H. Gene. 1998; 206: 273-282Crossref PubMed Scopus (61) Google Scholar). In addition to these inactive members of protease gene families, several phosphatase-like proteins that lack one or more residues critical for catalytic activity have been discovered (45Zhang Z.Y. Curr. Top. Cell. Regul. 1997; 35: 21-68Crossref PubMed Scopus (60) Google Scholar). Taken together with the present study, these results suggest th" @default.
• W2004671440 created "2016-06-24" @default.
• W2004671440 creator A5040010928 @default.
• W2004671440 creator A5075876819 @default.
• W2004671440 creator A5082020452 @default.
• W2004671440 date "1999-04-01" @default.
• W2004671440 modified "2023-10-16" @default.
• W2004671440 title "Glu300 of Rat Carboxypeptidase E Is Essential for Enzymatic Activity but Not Substrate Binding or Routing to the Regulated Secretory Pathway" @default.
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