Heterozygous mutations causing partial prohormone convertase 1 deficiency contribute to human obesity

Abstract

Null mutations in the PCSK1 gene, encoding the proprotein convertase 1/3 (PC1/3), cause recessive monogenic early onset obesity. Frequent coding variants that modestly impair PC1/3 function mildly increase the risk for common obesity. The aim of this study was to determine the contribution of rare functional PCSK1 mutations to obesity. PCSK1 exons were sequenced in 845 nonconsanguineous extremely obese Europeans. Eight novel nonsynonymous PCSK1 mutations were identified, all heterozygous. Seven mutations had a deleterious effect on either the maturation or the enzymatic activity of PC1/3 in cell lines. Of interest, five of these novel mutations, one of the previously described frequent variants (N221D), and the mutation found in an obese mouse model (N222D), affect residues at or near the structural calcium binding site Ca-1. The prevalence of the newly identified mutations was assessed in 6,233 obese and 6,274 lean European adults and children, which showed that carriers of any of these mutations causing partial PCSK1 deficiency had an 8.7-fold higher risk to be obese than wild-type carriers. These results provide the first evidence of an increased risk of obesity in heterozygous carriers of mutations in the PCSK1 gene. Furthermore, mutations causing partial PCSK1 deficiency are present in 0.83% of extreme obesity phenotypes.

FIG. 1.

Location of rare nonsynonymous mutations identified in the screening of 845 extremely obese subjects on the PC1/3 protein. A: The domains are depicted in the schematic representation of PC1/3. S, signal peptide; Pro, propeptide; C-Terminal, COOH-terminal domain. G593R has previously been described (12) and is included in this study as a control. B: Stereo representation of the PC1/3 model showing the protein backbone in gray cartoon representation, the dec-RVKR-CMK inhibitor in dark gray marking the active site cleft, and the two calcium ions in cyan. The sites of mutations found in this study are represented in green, and the previously described common variant N221D (15) and the N222D mutation found in an obese mouse model (14) are indicated in blue (prepared with PYMOL [DeLano Scientific LLC, www.pymol.org]).

FIG. 2.

PC1/3 activity is impaired in G226R, M125I, T175N, and N180S mutants. PC1/3 mutants were immunopurified from conditioned medium of transfected HEK293T and N₂A cells. Activity was determined using the fluorogenic substrate p-Glu-Arg-Thr-Lys-Arg-amino methylcoumarin and normalized for the amount of recombinant PC1/3. A: N₂A cells. B: HEK293T cells. EV, empty vector; WT, wild-type PC1/3. G593R recombinant PC1/3 serves as a negative control. Bars represent mean ± SD; n = 3–6 independent experiments conducted. *P < 0.05, **P < 0.01, ***P < 0.001.

FIG. 3.

Maturation and/or secretion are impaired in seven of eight PC1/3 mutants. Western blots of cell lysates and conditioned medium of transfected N₂A and HEK293T cells with EV (empty vector), WT (wild-type PC1/3), S325N, T175M, K26E, M125I, N180S, Y181H, G226R, T558A, or G593R using FLAG M2 for detection of recombinant PC1/3 proteins. ΔCt corresponds to COOH-terminal truncated PC1/3. G593R recombinant PC1/3 serves as a negative control.

FIG. 4.

The T175M mutation impairs N-glycosylation of PC1/3. Analysis was performed on transfected HEK293T cells. Absence (−) or presence (+) of endoglycosidase-F. Note the reduction in molecular weight of wild-type (WT) PC1/3 in the medium to the same apparent molecular weight of untreated (and treated) T175M mutant.