Glu300 of Rat Carboxypeptidase E Is Essential for Enzymatic Activity but Not Substrate Binding or Routing to the Regulated Secretory Pathway

Qian, Yimei; Varlamov, Oleg; Fricker, Lloyd D.

doi:10.1074/jbc.274.17.11582

Cited by 22 publications

(28 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Therefore, impaired sorting of CPE may prevent the proper targeting of proglucagon to granules. Our finding of impaired sorting of the E300Q mutant is at variance with a previous report that showed that it bound to its substrate and was sorted to granules normally in NIT3 cells (Qian et al 1999). It is not clear how CPE is missorted in N2a cells, since its C-terminal sorting domain (Fricker et al 1990) is intact in the E300Q mutant, as evidenced by our western blot results showing strong immunoreactivity in cell extracts using a C-terminal antibody.…”

Section: Discussioncontrasting

confidence: 56%

See 1 more Smart Citation

The sorting of proglucagon to secretory granules is mediated by carboxypeptidase E and intrinsic sorting signals

McGirr

Guizzetti

Dhanvantari

2013

Journal of Endocrinology

View full text Add to dashboard Cite

Proglucagon is expressed in pancreatic alpha cells, intestinal L cells and brainstem neurons. Tissue-specific processing of proglucagon yields the peptide hormones glucagon in the alpha cell and glucagon-like peptide (GLP)-1 and GLP-2 in L cells. Both glucagon and GLP-1 are secreted in response to nutritional status and are critical for regulating glycaemia. The sorting of proglucagon to the dense-core secretory granules of the regulated secretory pathway is essential for the appropriate secretion of glucagon and GLP-1. We examined the roles of carboxypeptidase E (CPE), a prohormone sorting receptor, the processing enzymes PC1/3 and PC2 and putative intrinsic sorting signals in proglucagon sorting. In Neuro 2a cells that lacked CPE, PC1/3 and PC2, proglucagon co-localised with the Golgi marker p115 as determined by quantitative immunofluorescence microscopy. Expression of CPE, but not of PC1/3 or PC2, enhanced proglucagon sorting to granules. siRNA-mediated knockdown of CPE disrupted regulated secretion of glucagon from pancreatic-derived alphaTC1-6 cells, but not of GLP-1 from intestinal cell-derived GLUTag cells. Mutation of the PC cleavage site K70R71, the dibasic R17R18 site within glucagon or the alpha-helix of glucagon, all significantly affected the sub-cellular localisation of proglucagon. Protein modelling revealed that alpha helices corresponding to glucagon, GLP-1 and GLP-2, are arranged within a disordered structure, suggesting some flexibility in the sorting mechanism. We conclude that there are multiple mechanisms for sorting proglucagon to the regulated secretory pathway, including a role for CPE in pancreatic alpha cells, initial cleavage at K70R71 and multiple sorting signals.

show abstract

Section: Discussioncontrasting

confidence: 56%

“…N2a wt cells were also stably transfected with the enzymatically inactive form of CPE, E300Q (Qian et al 1999) using Lipofectamine 2000. Stable transfectants were selected in 800 mg/ml G418, pooled and maintained in 400 mg/ml G418.…”

Section: Introductionmentioning

confidence: 99%

The sorting of proglucagon to secretory granules is mediated by carboxypeptidase E and intrinsic sorting signals

McGirr

Guizzetti

Dhanvantari

2013

Journal of Endocrinology

View full text Add to dashboard Cite

show abstract

“…The conversion of the putative active site Glu into Gln is not expected to substantially alter the folding of the protein; this is consistent with the observation that the mutant proteins are able to bind to the substrate affinity column despite the absence of activity. A previous study of Glu 300 in CPE (the equivalent residue) also found that the mutation to Gln eliminated activity but not substrate binding or routing to the regulated secretory pathway in a neuroendocrine cell line (48). In contrast, mutants that are severely misfolded are not properly FIG.…”

Section: Discussionmentioning

confidence: 99%

“…1). This Glu, which corresponds to Glu 270 in carboxypeptidase A and B and Glu 300 in CPE, has been previously been shown to be essential for enzyme activity but not substrate binding (48). Thus, this mutation would not be predicted to have a large impact on the structure of the protein.…”

mentioning

confidence: 99%

Characterization of the Enzymatic Properties of the First and Second Domains of Metallocarboxypeptidase D

Novikova

Eng

Lin

et al. 1999

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Carboxypeptidase D (CPD) contains three domains with homology to other metallocarboxypeptidases. To further characterize the various domains, we constructed a series of point mutants with a critical active site Glu of duck CPD converted to Gln. The proteins were expressed in the baculovirus system, purified to homogeneity, and characterized. Point mutations within both the first and second domains eliminated enzyme activity, indicating that the third domain is inactive toward dansyl-Phe-Ala-Arg. CPD removed only the C-terminal Lys or Arg from peptides, with the first domain more efficient toward Arg and the second domain more efficient toward Lys. Peptides containing Pro in the penultimate position were poorly cleaved by either domain. Cleavage of a peptide with Ala in the penultimate position was most efficient, with the relative order Ala > Met > Ser, Phe > Tyr > Trp > Thr > Gln, Asp, Leu, Gly Ͼ Ͼ Ͼ Ͼ Pro for CPD with both domains active. There were only minor differences between the first and the second domains regarding the influence of the penultimate amino acid. The first domain was optimally active at pH 6.3-7.5, whereas the second domain was optimally active at pH 5.0 -6.5. Thus, the first and second carboxypeptidase domains have complementary enzyme activities. Furthermore, the finding that CPD with both domains active shows a broad activity to a wide range of substrates is consistent with a role for this enzyme in the processing of many proteins that transit the secretory pathway.

show abstract

“…Substitution of this Glu residue with a Gln abolishes the activity of CPE, but does not affect the binding of synthetic peptide substrates (Qian et al, 1999). If cCPZ functions as an enzyme, one would expect that loss of its catalytic activity should manifest itself by an inability to induce Pax3 expression.…”

Section: ---------------------------------------------mentioning

confidence: 99%

Carboxypeptidase Z (CPZ) modulates Wnt signaling and regulates the development of skeletal elements in the chicken

Moeller¹,

Swindell

Kispert

et al. 2003

Development

View full text Add to dashboard Cite

Carboxypeptidase Z (CPZ) is a secreted Zn-dependent enzyme whose biological function is largely unknown. CPZ has a bipartite structure consisting of an N-terminal cysteine-rich domain (CRD) and a C-terminal catalytic domain. In the early chicken embryo CPZ is initially expressed throughout the somites and subsequently becomes restricted to the sclerotome. To initiate a functional analysis of CPZ, a CPZ producing retroviral vector was applied to the presomitic mesoderm at the level of the future wing. This resulted in a loss of the scapular blade and of rostral ribs. Such dysmorphogenesis is preceded by ectopic Pax3 expression in the hypaxial part of the dermomyotome,a region from which the blade of the scapula normally derives. A mutant CPZ,lacking a critical active site glutamate, fails to induce Pax3expression and does not cause skeletal defects. The induction of Pax3, a Wnt-responsive gene in somites, and the presence of a CRD prompted us to examine whether CPZ affects Wnt signaling. In an in vitro assay we found that CPZ, but not its inactive mutant form, enhances the Wnt-dependent induction of the homeobox gene Cdx1. In addition,immunoprecipitation experiments suggest that the CRD of CPZ acts as a binding domain for Wnt. Taken together these data provide the first evidence for CPZ playing a role in Wnt signaling.

show abstract

Glu300 of Rat Carboxypeptidase E Is Essential for Enzymatic Activity but Not Substrate Binding or Routing to the Regulated Secretory Pathway

Cited by 22 publications

References 46 publications

The sorting of proglucagon to secretory granules is mediated by carboxypeptidase E and intrinsic sorting signals

The sorting of proglucagon to secretory granules is mediated by carboxypeptidase E and intrinsic sorting signals

Characterization of the Enzymatic Properties of the First and Second Domains of Metallocarboxypeptidase D

Carboxypeptidase Z (CPZ) modulates Wnt signaling and regulates the development of skeletal elements in the chicken

Contact Info

Product

Resources

About