Small-intestinal dysfunction accompanies the complex endocrinopathy of human proprotein convertase 1 deficiency

Abstract

We have previously described the only reported case of human proprotein convertase 1 (PC1) deficiency, in a female (Subject A) with obesity, hypogonadism, hypoadrenalism, and reactive hypoglycemia. We now report the second case of human PC1 deficiency (Subject B), also due to compound heterozygosity for novel missense and nonsense mutations. While both subjects shared the phenotypes of obesity, hypoadrenalism, reactive hypoglycemia, and elevated circulating levels of certain prohormones, the clinical presentation of Subject B was dominated by severe refractory neonatal diarrhea, malabsorptive in type. Subsequent investigation of Subject A revealed marked small-intestinal absorptive dysfunction, which was not previously clinically suspected. We postulate that PC1, presumably in the enteroendocrine cells, is essential for the normal absorptive function of the human small intestine. The differences in the nature and severity of presentation between the two cases cannot readily be explained on the basis of allelic heterogeneity, as the nonsense and missense mutations from both subjects had comparably severe effects on the catalytic activity of PC1. Despite Subject A's negligible PC1 activity, some mature ACTH and glucagon-like peptide 17-36(amide) were detectable in her plasma, suggesting that the production of these hormones, at least in humans, does not have an absolute dependence on PC1. The presence of severe obesity and the absence of growth retardation in both subjects contrast markedly with the phenotype of mice lacking PC1 and suggest that the precise physiological repertoire of this enzyme may vary between mammalian species.

Figure 1

PC1 mutations in Subject B. (a) Bidirectional cycle sequencing of genomic DNA showed Subject B to be compound heterozygous for Ala213del (A213Δ; deletion of GCA or CAG) and Glu250stop (937G→T), inherited from her mother and her father, respectively. (b) A linear model of PC1, showing the domains and sites of mutations found in Subjects A and B.

Figure 2

Subject A secreted mature gastrin, but her elevated postprandial plasma progastrin level suggested some impairment of processing. (a) Schematic of sequential cleavage and C-terminal modification of gastrin forms. G17 is mature gastrin. (b and c) Plasma progastrin (b) and total amidated gastrin (c) were measured by RIA using antisera L289 and L2, respectively, in the subject (squares) and ten healthy controls (triangles), before and at 20-minute intervals after the start of a high-fat and -carbohydrate test meal. Mature gastrin, G17, was also measured in the subject, using the highly specific antiserum L6 (circles). Control data are mean ± SE.

Figure 3

Subjects A’s plasma contained products that normally result from PC1-mediated proglucagon processing. (a) PC1 in intestinal L cells cleaves proglucagon to glicentin, oxyntomodulin, and GLP-1 and -2, while in islet A cells PC2 forms glucagon, 9-kDa peptide, and major proglucagon fragment (MPGF). GRPP, glicentin-related pancreatic peptide; IP, intervening peptide. (b) Reverse-phase HPLC of postprandial plasma with RIA of eluted amidated C-terminal GLP-1 revealed the continued presence of mature GLP-1^7-36amide. ↓, GLP-1 standards. (c) GLP-2 also was detectable in postprandial plasma using size-exclusion gel chromatography and RIA of eluted mid-sequence GLP-2 (mid–GLP-2). (d) In response to food, Subject A’s apparently normal fasting plasma levels of glicentin, oxyntomodulin, and glucagon rose abnormally high. Size-exclusion gel chromatography with RIA (antiserum 4304) of eluted mid-sequence glucagon (present in glicentin, oxyntomodulin, and glucagon) was applied to plasma from Subject A (top) and five pooled controls (bottom) during fasting (left) and 1 hour after food intake (right). Oxyn., oxyntomodulin; K_diss, coefficient of distribution; ↓, standards; IR, immunoreactivity.

Figure 4

Recombinant PC1-Gly593Arg (PC1-G593R) and PC1-Ala213del (PC1-A213Δ) were catalytically inactive. (a) Western blot analysis of CHO-K1 and βTC3 cells transfected with empty vector (C) or WT PC1, PC1-G593R, or PC1-A213Δ cDNA. Recombinant PC1 was detected with an anti-FLAG antibody. ΔCT, C-terminally truncated PC1. (b) Fluorogenic assay of PC1 proteolytic activity using anti-FLAG–immunopurified PC1 from lysates of βTC3 cells transfected as in a. Open circles, empty vector; filled diamonds, WT PC1 cDNA; open squares, PC1-G593R cDNA; open triangles, PC1-A213Δ cDNA. Activity was normalized for input of recombinant protein. (c) Co-immunoprecipitation of the propeptide of WT PC1 and PC1-A213Δ. Radiolabeled cell lysates (30-minute pulse, 1-hour chase) were immunoprecipitated with anti-FLAG antibody as in b. The lower part of the gel was exposed longer to visualize the propeptide.

Figure 5

Transfection of a PC1 minigene containing the splice-site variant resulted in undetectable full-sized PC1 protein and catalytic activity. (a) Western blot (left and middle panels) and immunoprecipitation analysis (right panel) of HEK-293T cells transfected with empty vector (C) or PC1 minigene containing either WT or splice-site variant (SSV) sequences. Cells used for immunoprecipitation were pulse-labeled for 1 hour. Endo F, endoglycosidase F. (b) Fluorogenic assay of PC1 proteolytic activity using PC1 immunopurified from lysates of αTC1-6 cells transfected as in a. Open circles, empty vector; filled diamonds, PC1 minigene; open squares, PC1 splice-site variant minigene.

Figure 6

Physiologically regulated, qualitatively normal POMC products were present in Subject A’s plasma. (a) POMC is the precursor to multiple distinct peptides. N-POMC, N-terminal fragment of POMC; JP, joining peptide; αMSH, α-melanocyte stimulating hormone; βLPH, β lipotropin. (b) Concentrations of plasma cortisol (diamonds; ng/ml), ACTH (squares; pg/ml), and POMC (triangles; × 100 U/ml) were determined over 24 hours (0900 hours to 0900 hours) in Subject A. Immediately after the 0900-hours sample on day 2, a 100-μg i.v. bolus of CRH was administered. (c) HPLC of plasma sampled 20 minutes after CRH administration, with RIA of eluted C-terminal ACTH, showed peaks of phosphorylated (ACTH-p) and nonphosphorylated ACTH 1-39 (ACTH), confirming the results of direct plasma assays. Neither CLIP (a product of PC2 activity) nor immunoreactive peptides of abnormal size were present, indicating that POMC processing was of normal specificity. ↓, standards. (d) Size-exclusion gel chromatography of early-morning plasma with β endorphin RIA (triangles) and POMC IRMA (circles) of eluted peptides showed POMC and β lipotropin to be present but not β endorphin, which is a product of processing of POMC by PC2. Results are expressed as percentages of the total immunoreactivity eluted. ↓, standards.