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CELA2A mutations predispose to early-onset atherosclerosis and metabolic syndrome and affect plasma insulin and platelet activation

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Factors that underlie the clustering of metabolic syndrome traits are not fully known. We performed whole-exome sequence analysis in kindreds with extreme phenotypes of early-onset atherosclerosis and metabolic syndrome, and identified novel loss-of-function mutations in the gene encoding the pancreatic elastase chymotrypsin-like elastase family member 2A (CELA2A). We further show that CELA2A is a circulating enzyme that reduces platelet hyperactivation, triggers both insulin secretion and degradation, and increases insulin sensitivity. CELA2A plasma levels rise postprandially and parallel insulin levels in humans. Loss of these functions by the mutant proteins provides insight into disease mechanisms and suggests that CELA2A could be an attractive therapeutic target.
Human WT and mutant CELA2A structures and functions a, Western blot image showing His-WT-, His-p.D121N-, His-p.T70M-, His-c.639+1G>C- and His-p.L85M-CELA2A expressed in HEK293T cells from cell lysate (bottom) and secreted supernatant (top). An extra band at 75 kDa is present in secreted WT-CELA2A and p.L85M-CELA2A, verified as CELA2A protein by proteomic analysis (n = 3 replicated experiments). b, Ribbon schematics of the catalytic triad within the crystal structures of WT- and p.D121N-CELA2A. For the WT, Asp121 and the hydrogen bonds between its β-carboxyl group and N delta 1 of His73 are shown. For p.D121N-CELA2A, p.D121N substitution is shown, which disrupts hydrogen bonds. c, In vitro elastase activity of purified WT and different mutant CELA2A proteins (means ± s.e.m.; n = 3 replicated experiments). Significance was determined by one-way ANOVA with a 95% confidence interval (CI) (***P = 0.001). d, Dot plot showing the elastase activity of WT-CELA2A co-expressed with different mutant CELA2A proteins (means ± s.e.m; n = 2 replicated experiments). Significance was determined by one-way ANOVA with a 95% CI (****P < 0.0001). e, Western blot analysis of A1AT treated with WT-, p.D121N-, p.T70M-, c.639+1G>C- and p.L85M-CELA2A. Red color depicts CELA2A, green depicts the A1A2 and yellow depicts the merging of the two, indicating their complex formation. The 75-kDa bands of WT- and p.L85M-CELA2A bound to A1AT are shown (lanes 2 and 6). The 75-kDa band is absent in all other mutant CELA2A proteins (lanes 3–5) (n = 3 replicated experiments).
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Mouse and human Cela2a tissue expression, human CELA2A plasma levels and activities a,b, Cela2a messenger RNA (a; means ± s.e.m.) and protein levels (b) in different mouse tissues (n = 6 mice). c, Immunohistochemical staining of human tissues using CELA2A-specific antibody. Controls without primary antibodies are also shown (n = 1). Scale bars: 100 μm. d, Western blot analysis of CELA2A in human serum (75- and 25-kDa Cela2a bands). Both bands are absent in the parallel western blot performed on human serum using CELA2A-specific antibody preblocked with rCela2a (n = 2 replicated experiments). The whole blot is shown in Supplementary Fig. 7. e,f, Total plasma CELA2A levels (e) and elastase activity (f) (means ± s.e.m.) measured in p.D121N carriers versus non-carriers, measured by validated ELISA assay (n = 7 in each group). Significance was determined by two-tailed Student’s t-test (**P < 0.01). g–i, Baseline and postprandial plasma CELA2A (g), insulin (h) and C-peptide (i) levels (means ± s.e.m.; n = 8). Significance was determined by two-tailed Student’s t-test (**P = 0.0009; **P < 0.0001 and **P = 0.0006, respectively). j,k, Correlation of baseline and postprandial plasma insulin (j) and C-peptide (k) with plasma CELA2A in random healthy subjects (n = 8; age: 20–30 years), presented as values. Correlation coefficients for j and k were calculated using GraphPad Prism 8. The dashed lines denote the 95% confidence area of the graph. l,m, Plasma glucose and CELA2A levels (means ± s.e.m.) during hyperglycemic clamps (l) and OGTT (m) in obese non-diabetic subjects (n = 5).
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Insulin secretion, degradation and sensitivity of WT-CELA2A and p.D121N-CELA2A proteins a,b, Insulin (a) and C-peptide (b) secretion of rat islets in response to WT-CELA2A (50 nM), p.D121N-CELA2A or vehicle at 2.5 and 9 mM glucose concentrations, represented as violin plots showing mean, minimum and maximum values (n = 4 in two replicated experiments). **P < 0.01 by one-way ANOVA. KCl was used as a positive control and test of viability. c, Calcium transient representations of INS-1 cells treated with WT- versus p.D121N-CELA2A. d, AUC of Ca²⁺ transient in the cells shown in c, represented as dot plots (means ± s.e.m.; n = 3 biologically independent experiments). Panels c and d show greater calcium transient in WT-CELA2A-treated cells. Significance in d was determined by one-way ANOVA with a 95% CI (***P < 0.0001). e, Insulin content (means ± s.e.m) in the media of rat islets treated with WT- and p.D121N-CELA2A for 48 h, represented as dot plots (n = 3 replicated experiments). Significance was determined by two-sided Student’s t-test with a 90% CI (*P < 0.05). f, UPLC analysis of insulin digestion by WT-, p.D121N-, p.T70M- and p.L85M-CELA2A (n = 2 replicated experiments). Arrows show fragmented insulin. g, Western blot analysis of insulin/mammalian target of rapamycin signaling pathways in 3T3L1 cells treated with insulin, WT- or p.D121N-CELA2A and predigested insulin with WT- or p.D121N-CELA2A (n = 2 biologically independent experiments; quantification in Supplementary Fig. 6; whole blot in Supplementary Fig. 7). IRS and pIRS denote insulin receptor substrate and its phosphorylation, respectively. pAKT denotes phosphorylated AKT, also known as protein kinase B (PKB).
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Articles
https://doi.org/10.1038/s41588-019-0470-3
1Department of Internal Medicine, Yale School of Medicine, New Haven, CT, USA. 2Center for Exocrine Disorders, Department of Molecular and Cell
Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, MA, USA. 3Department of Medicine, NYU Medical Center, New York,
NY, USA. 4Department of Genetics, Yale School of Medicine, New Haven, CT, USA. 5Laboratory of Human Genetics and Genomics, The Rockefeller
University, New York, NY, USA. 6Department of Pathology, Yale School of Medicine, New Haven, CT, USA. *e-mail: arya.mani@yale.edu
Metabolic syndrome is a cluster of inherited risk factors
for coronary artery disease (CAD)13, which in out-
lier kindreds with early-onset CAD may be caused by
single-gene mutations46. In this study, we present a cohort of 30
North European index cases with early-onset CAD and meta-
bolic syndrome. Combined linkage and gene burden analyses led
to the identification of multiple independent mutations in
CELA2A, which encodes the chymotrypsin-like elastase family
member 2A (CELA2A).
CELA2A was primarily known as an ‘exocrine’ pancreatic elas-
tase that preferentially cleaves -acetyl--alanyl--alanyl--alanine/
proline methyl-ester7 and forms a sodium dodecyl sulfate (SDS)-
resistant complex with alpha-1-antitrypsin (A1AT)8. The physi-
ological function of CELA2A outside the exocrine pancreas was
not known. Here, we characterize the CELA2A protein invitro and
invivo, and explore the effects of human mutations on its diverse
metabolic functions. Using systems biology, we discovered that
CELA2A is a circulating protein that impacts diverse biological pro-
cesses, including insulin secretion, degradation and sensitivity. Our
analyses show that impaired regulation of plasma insulin is a major
consequence of disease-inducing CELA2A mutations. The potential
to exploit disease pathways makes CELA2A an appealing target for
treating diabetes and its complications.
Results
Clinical characterization of individuals and families with early-
onset CAD and metabolic syndrome. We recruited 30 index cases
with early-onset CAD (age of onset: at or before 30 years in men
and 35 years in women) and extended their kindreds. This led to
the identification and recruitment of a multiplex kindred with
25 affected individuals that we named CAD-2001 (Fig. 1a). The
index case was an American female of European ancestry with an
extensive family history of CAD, and first ST elevation myocardial
infarction at age 28 years (arrow). Her CAD risk factors included
hypertension (HTN), type 2 diabetes (T2D), hypertriglyceridemia
(HTG) and obesity. She underwent a coronary artery angiography
and percutaneous intervention of two major coronary arteries.
Among 53 extended blood relatives of the index case, 25 were diag-
nosed with early-onset CAD (a myocardial infarction diagnosis by
enzyme and electrocardiogram, angiographic diagnosis of CAD, or
sudden cardiac death) with a median age of 43 years, and 11 had
died from CAD (mean age of death: 52 years). All affected individu-
als traced their ancestry to a common ancestor, and male-to-female
transmission of the phenotype was present.
Detailed clinical data were available for all 11 living family mem-
bers with CAD, two younger family members (mean age: 30 years)
with unknown CAD status, and 12 living unaffected family mem-
bers (Supplementary Table 1). All affected individuals met the
National Cholesterol Education Program criteria for metabolic syn-
drome with surprisingly homogeneous risk factors, including mark-
edly elevated triglycerides (mean 287.1 mg dl1; Nl < 150 mg dl1),
HTN, low high-density lipoprotein (HDL; mean 35.1 mg dl1;
Nl > 50 mg dl1) and T2D (fasting blood glucose > 126 mg dl1 or
on glucose-lowering drugs). In contrast, all 12 unaffected family
members had normal triglycerides (mean: 100.5 mg dl1) and near-
normal HDL levels, and none had T2D. The two younger family
members with unknown CAD status had both HTG and HTN.
The familial clustering and pattern of inheritance of these clinical
CELA2A mutations predispose to early-onset
atherosclerosis and metabolic syndrome and
affect plasma insulin and platelet activation
Fatemehsadat Esteghamat 1, James S. Broughton1, Emily Smith1, Rebecca Cardone1, Tarun Tyagi1,
Mateus Guerra 1, András Szabó2, Nelson Ugwu 1, Mitra V. Mani1, Bani Azari1, Gerald Kayingo1,
Sunny Chung1, Mohsen Fathzadeh1, Ephraim Weiss3, Jeffrey Bender1, Shrikant Mane4,
Richard P. Lifton5, Adebowale Adeniran6, Michael H. Nathanson1, Fred S. Gorelick1, John Hwa1,
Miklós Sahin-Tóth 2, Renata Belfort-DeAguiar1, Richard G. Kibbey1 and Arya Mani 1,4*
Factors that underlie the clustering of metabolic syndrome traits are not fully known. We performed whole-exome sequence
analysis in kindreds with extreme phenotypes of early-onset atherosclerosis and metabolic syndrome, and identified novel loss-
of-function mutations in the gene encoding the pancreatic elastase chymotrypsin-like elastase family member 2A (CELA2A).
We further show that CELA2A is a circulating enzyme that reduces platelet hyperactivation, triggers both insulin secretion and
degradation, and increases insulin sensitivity. CELA2A plasma levels rise postprandially and parallel insulin levels in humans.
Loss of these functions by the mutant proteins provides insight into disease mechanisms and suggests that CELA2A could be
an attractive therapeutic target.
NATURE GENETICS | VOL 51 | AUGUST 2019 | 1233–1243 | www.nature.com/naturegenetics 1233
Content courtesy of Springer Nature, terms of use apply. Rights reserved
... This connection between diabetes onset and dysregulated protease production provides additional evidence that acinar cell secretions may play a role in the maintenance of beta cell health. Similarly, pancreatic elastase (encoded by CELA2A) is known to enhance insulin signaling by both promoting insulin secretion and mediating degradation (82). In the presence of CELA2A gene mutations and therefore nonfunctional CELA2A protein, dysregulated insulin secretion, hyperglycemia, and the eventual onset of T2D results (82,95). ...
... Similarly, pancreatic elastase (encoded by CELA2A) is known to enhance insulin signaling by both promoting insulin secretion and mediating degradation (82). In the presence of CELA2A gene mutations and therefore nonfunctional CELA2A protein, dysregulated insulin secretion, hyperglycemia, and the eventual onset of T2D results (82,95). Without this essential exocrine-derived protein, proper insulin signaling is unachievable. ...
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