Biochemical and cell biological properties of the human prohormone convertase 1/3 Ser357Gly mutation: a PC1/3 hypermorph

Abstract

Satiety and appetite signaling are accomplished by circulating peptide hormones. These peptide hormones require processing from larger precursors to become bioactive, often by the proprotein convertase 1/3 (PC1/3). Several subcellular maturation steps are necessary for PC1/3 to achieve its optimal enzymatic activity. Certain PC1/3 variants found in the general population slightly attenuate its enzymatic activity and are associated with obesity and diabetes. However, mutations that increase PC1/3 activity and/or affect its specificity could also have physiological consequences. We here present data showing that the known human Ser357Gly PC1/3 mutant (PC1/3(S357G)) represents a PC1/3 hypermorph. Conditioned media from human embryonic kidney-293 cells transfected with PC1/3(WT) and PC1/3(S357G) were collected and enzymatic activity characterized. PC1/3(S357G) exhibited a lower calcium dependence; a higher pH optimum (neutral); and a higher resistance to peptide inhibitors than the wild-type enzyme. PC1/3(S357G) exhibited increased cleavage to the C-terminally truncated form, and kinetic parameters of the full-length and truncated mutant enzymes were also altered. Lastly, the S357G mutation broadened the specificity of the enzyme; we detected PC2-like specificity on the substrate proCART, the precursor of the cocaine- and amphetamine regulated transcript neuropeptide known to be associated with obesity. The production of another anorexigenic peptide normally synthesized only by PC2, αMSH, was increased when proopiomelanocortin was coexpressed with PC1/3(S357G). Considering the aberrant enzymatic profile of PC1/3(S357G), we hypothesize that this enzyme possesses unusual processing activity that may significantly change the profile of circulating peptide hormones.

Figure 1.

Serine 357 is conserved in PC1/3. A, Alignment of PC1/3s from several species shows that serine in position 357 is highly conserved. Amino acid sequences from the National Center for Biotechnology Information database used were as follows: Homo sapiens (NP_000430.3), Mus musculus (NP_038656.1), Rattus norvegicus (NP_058787.1), Danio rerio (NP_001131134.1), Gallus gallus (XP_003643108.1), Chrysemys picta bellii (XP_005284059.1), Xenopus tropicalis (XP_002933476.2), Pseudopodoces humilis (XP_005533963.1), and Tursiops truncatus (XP_004321922.1). For multiple PC1/3 sequence alignment, we used ClustalW2 web software (21). Asterisks show conserved residues among species. B, The PC1/3^S357G model is based on the known furin structure; this model was obtained courtesy of Than et al (33). The catalytic domain is represented in purple and the P domain in light blue. The catalytic triad (D167–H208-S382) is shown in black spheres and the oxyanion hole N309 with a black stick. G357 is shown with orange spheres. Two calcium ions are shown as yellow spheres. A tetrapeptide substrate is depicted in red.

Figure 2.

PC1/3^S357G exhibits increased autoconversion to the 66-kDa truncated form. Samples from conditioned media obtained from transfected Neuro2A (A) or HEK293 (B) cells were collected and analyzed by Western blotting (top panels) and enzymatic assay (bottom panels). Cells were transfected with vectors encoding full-length PC1/3^WT, full-length PC1/3^S357G, or empty vector. The antiserum used for Western blotting recognizes all PC1/3 forms. Enzymatic activity assay shows AMC release with respect to time. The lane on the right shows recombinant mPC1 protein, used as a positive control. C, Neuro2A cells were transfected with vectors encoding full-length PC1/3^WT or full-length PC1/3^S357G, radiolabeled, and cell extracts and media subjected to immunoprecipitation. A 20-minute pulse of medium containing radioactive methionine followed by a 2-hour chase with medium containing cold methionine is shown. ProPC1/3 is detected at 94 kDa, PC1/3 at 87 kDa, and the truncated form of PC1/3 at 66 kDa.

Figure 3.

PC1/3^S357G shows exponential enzyme kinetics and alterations in pH and calcium requirements. A, Samples from conditioned media HEK293 cells were collected and analyzed by Western blotting and enzymatic assay. HEK293 cells were transfected with vectors encoding the PC1/3^WT truncated form or the PC1/3^S357G truncated form. The conditioned media were used at pH 5.5 and 2 mM calcium. An enzymatic activity assay shows AMC release with respect to time. B, For pH experiments, the buffer used was 100 mM Bis-Tris + 100 mM sodium acetate, and the different pHs were obtained using 5 N acetic acid. Calcium was fixed at 2 mM in all buffers. C, For calcium experiments, 0.9 mM EDTA (final concentration) was added to conditioned media for neutralization of the calcium present in the medium. The calcium curve was then made in regular reaction buffer at pH 5.5. AMC release per minute was determined at 60 minutes of enzymatic reaction. A′, B′, and C′, Expression levels of truncated forms of PC1/3 (Western blotting). Samples were obtained from the experiments shown in panels A, B, and C, respectively.

Figure 4.

PC1/3^S357G shows increased tolerance to inhibitor peptide. Samples from conditioned media of HEK293 cells were collected and analyzed by Western blotting and enzymatic assay. The HEK293 cells were transfected with vectors encoding either the PC1/3^WT truncated form (A) or the PC1/3^S357G truncated form (B). A and B, Enzymatic activity assays show AMC release with respect to time. Samples were preincubated with LLRVKR at different concentrations prior to adding the fluorogenic peptide substrate (pERTKR-AMC). The standard buffer containing pH 5.5 and 2 mM calcium was used. C, Expression levels of the truncated forms of PC1/3 used in this experiment (Western blotting of parallel reaction mixtures).

Figure 5.

A proCART fusion protein is processed more extensively in Neuro2a cells by PC1/3^S357G. Neuro2A/proCART stable cells were transfected with cDNAs encoding PC1/3^WT or PC1/3^S357G full-length (A) or their respective truncated forms (B). The negative control used was empty vector and the positive control was an mPC2 vector. Conditioned media were used for Western blotting analysis. Upper panels show the expression levels of proCART-EGFPm forms by Western blotting using an antibody against GFP. Bottom panels show the expression levels of PC1/3 by Western blotting. C, proCART-EGFP processing scheme. WB, Western blot.

Figure 6.

POMC and, to a lesser extent, proglucagon are more extensively processed to α-MSH and glucagon by PC1/3^S357G. GH4C1 cells were cotransfected with either POMC or proglucagon with the PC1/3 full-length enzymes indicated. Cell extracts and conditioned media were analyzed for α-MSH (A), ACTH (B), or glucagon (D). Data were analyzed using a one-way ANOVA followed by a post hoc Bonferroni test. *, P <.05; **, P < .01; ***, P < .001. Panels C and E show POMC (C) and proglucagon (E) processing schema.

Figure 7.

PC1/3^Q665E+S690T and PC1/3^WT show identical enzymatic activity in both Neuro2A and HEK293 cells. Samples of conditioned media obtained from transfected Neuro2A (A) or HEK293 (B) cells were collected and analyzed by Western blotting and enzymatic assay. Cells were transfected with vectors encoding full-length PC1/3^WT, full-length PC1/3^Q665E+S690T, or empty vector. The antiserum used for Western blotting recognizes all PC1/3 forms. Enzymatic activity depicts AMC release with respect to time. A′ and B′, Expression levels of full-length PC1/3^WT or PC1/3^Q665E+S690T (Western blotting). Samples correspond to the experiments shown in panels A and B, respectively.

Figure 8.

Glycine specificity of the Ser357Gly mutation: Ser357Ala decreases PC1/3 activity, whereas the Asn357Gly mutation increases PC2 activity. A, Samples of conditioned media obtained from transfected HEK293 cells were analyzed by Western blotting and enzymatic assay. Cells were transfected with vectors encoding full-length PC1/3^WT, full-length PC1/3^S357A, or empty vector. The antiserum used for Western blotting recognizes all PC1/3 forms. Enzymatic activity depicts AMC release with respect to time. Panel A′, Expression levels of full-length PC1/3^WT or PC1/3^S357A (Western blotting); samples correspond to the experiments shown in panel A. Panel B, Samples of conditioned media obtained from transfected Neuro2A cells were collected and analyzed by Western blotting and enzymatic assay. Cells were transfected with vectors encoding either full-length PC2^WT, full-length PC2^N357G, or empty vector. The antisera combination used for Western blotting recognizes all PC2 forms. Enzymatic activity depicts AMC release with respect to time. Panel B′, Expression levels of full-length PC2^WT or PC2^N357G (Western blotting); media and lysate samples correspond to the experiments shown in panel B.