Functional And Mechanical Studies Of Secretagogin In Intestinal Hormone Secretion And Inflammatory Bowel Disease

Inflammatory bowel disease（IBD）is a general term for immune diseases involving chronic inflammation of the digestive tract.The etiology of IBD is complex and it is thought that by both environment triggers and genetic predisposition are required.IBD follows two broad phenotypic presentations,which are referred to as Crohn’s disease（CD）and ulcerative colitis（UC）.IBD is rare in non-industrialized societies and is becoming increasingly prevalent as with the advent of industrialization all over the world.The prevalence of IBD in the United States has been estimated at about 0.5-1.3%of the population,which is similar to the estimates in western European countries;areas of the world that are undergoing rapid industrialization are quickly approaching these figures.A recent study shows that there are more than 6.8 million people affected by IBD in the world.IBD being a chronic and incurable condition can cause great morbidity,as well as heavy economic burden not only to the patient but to society as a whole.There are various environmental factors associated with IBD risk,including westernstyle diet,air pollution,antibiotics usage,microbial exposures,mode of birth,to name a few.Regarding the studies of genetic factors,high throughput genomic research tools,such as Genome-wide association studies（GWAS）,have been used to try to identify the genetic contribution to IBD.To date,more than 200 loci have been identified as IBD associated risk genes.However,most of these gene variants are intergenic and are associated with modest risk,with odds ratios below 2（only exceptions are the NOD2 and IL23R risk alleles）.In aggregate,GWAS only accounts for 25-30%of the genetic risk,thus leaving a significant proportion of heritability that is currently unexplained.To try to close that gap,pediatric presentations of IBD have been more recently studied.Approximately 25%of IBD cases present during childhood.The studies of IBD risk genes in pediatric population focus on monogenic Mendelian traits.Over 50 loci have been confirmed in pediatric IBD and additional gene discovery,especially in early onset IBD patients,remains an area of active investigation.In this study,we identified a consanguineous family with three out of five children affected by aggressive,early onset ulcerative colitis.We employed multiple genomic research methods,including linkage analysis,whole exon sequencing,whole genomic sequencing,and Sanger sequencing,to study the genetic basis of the disease in this family.We found that a very rare（MAF<1%）homozygous nonsynonymous mutation in SCGN gene（rs376721140;NM₀06998.3:c.230G>A;p.Arg77His）was shared by the three patients but not by their healthy siblings.Both parents were identified as heterozygote carriers,while one of the unaffected siblings had no mutations and the other was a heterozygote carrier.Secretagogin（SCGN）,encoded by SCGN gene,is a highly conserved hexa EF-hand Ca2+ sensor,which is mainly expressed in neuroendocrine cells in pancreas,brain and endocrine cells of the gut（enteroendocrine cells or EECs）.SCGN is the most abundant protein in pancreatic β cells and initial studies suggested that SCGN positively affects insulin synthesis and secretion by regulating actin dynamics as well as direct binding to insulin molecule.Other studies also reported that type Ⅱ diabetes patients had less SCGN expression compared to healthy individuals and that SCGN regulated pancreaticβ cell specification by protecting Pdxl from proteasomal degradation.In addition to the regulation of insulin synthesis and secretion in pancreatic β cells,SCGN expressed in brain neurons also participates in the release processes of adrenergic hormone（Corticotropin-releasing hormone,CRH）and matrix metalloprotease-2（MMP-2）.However,an overall mechanism for the reported functions of SCGN was not evident from these initial studies.An important breakthrough was the discovery that SCGN binds to SNAP-25,an important component of the Soluble Nethylmaleimide-sensitive-factor attachment receptor（SNARE）complex,which participates in various cellular events by mediating the membrane fusion process,for instance,during neurotransmitter and hormone release,autophagy,cell division,cell migration,etc.SCGN may function as one of the regulatory proteins of the SNARE complex via interacting with SNAP-25,but a detailed interaction mechanism remains unclear.Hence,we investigated the specific functional mechanism of SCGN during hormone release process and the potential effect of the IBD-associated mutation we had found.To study the relationship between SCGN and IBD onset in depth,firstly,we studied the expression pattern of SCGN in the colon of healthy individuals and our patients.Immunohistochemistry staining indicated that SCGN was equally expressed in both unaffected individuals and the probands,and was detectable in triangular-shaped intestinal epithelial cells,consistent with enteroendocrine cells（EECs）.These cells had comparable morphology by light and electron microscopy.Next,using immunofluorescent staining for markers of EECs as well as gut neurons we found that only a subpopulation of EECs and gut neurons were SCGN positive.To further explore the mechanism by which SCGN exerts its physiological functions in the intestine,we developed a cellular model of SCGN deficiency in an enteroendocrine cell of mouse origin,known as STC-1.These cells secrete GLP-1,a gut hormone,in response to nutrient exposure in the growth media.Using CRISPR/Cas9,we first generated STC-1 cells that were KO for SCGN and then rescued these cells to express human wild-type（WT）or mutant（p.R77H）SCGN.To further understand the function of SCGN in hormone secretion,we studied the interactome of SCGN in the EECs by mass spectrometry.Our results indicated that SCGN interacts with proteins related to secretion,such as SNAP-25,SNAP-23,and Doc2a,consistent with previous reports in other cells.In this study,we focused on the interaction between SCGN and SNAP-25.Our collaborator was able to identify the direct binding sites of SCGN and SNAP-25 through X-ray crystal diffraction.Then we designed two mutants of SCGN（F225A and M229A）based on the structure information,which should suppress the interaction with SNAP-25,and developed STC-1 cells expressing these mutant proteins through rescue SCGN KO clones.Next,we studied the binding ability to SNAP-25 of SCGN mutants（F225A,M229A and R77H）by immunoprecipitation.The experiment results indicated that F225A and M229A mutants impair the binding ability to SNAP-25;interestingly R77H had no impairment,as this residue is far away from the SNAP-25 interacting domain.Then,we established a fatty-acid-stimulated in vitro Glucagon-like peptide-1（GLP-1）release assay using STC-1 cells.We employed 100 μM Docosahexaenoic acid（DHA）to stimulate parental,SCGN KO or rescue STC1 cell lines and analyzed secreted hormone concentration in the medium by ELISA.The results showed that,SCGN deficient STC-1 cells significantly lost hormone secretion ability compared to parental STC-1 cells.We observed that the SCGN mutations that impaired SNAP-25 binding,F225A and M229A,as well as the IBD associated mutant,R77H,disturbed GLP-1 secretion and the normal membranous localization of SNAP-25 by immunofluorescent staining.In aggregate,our experiment results confirmed that SCGN controls optimal GLP-1 secretion by directly binding to SNAP-25 and regulating its subcellular localization.The nature of how R77H impairs secretion remains unaccounted for by our current structural understanding of how SCGN binds to SNAP-25.Next,we developed two Scgn knockout（KO）mouse models using CRISPR/Cas9 technology to investigate the role of SCGN in mouse models of IBD.First,Scgn deficient mice did not develop spontaneous colitis,as determined by histological analysis,as well as mRNA expression of inflammation markers via RT-qPCR.Then we used the dextran sulfate sodium（DSS）acute colitis model to study the susceptibility of Scgn deficient mice to this model.Our results indicated that Scgn deficient mice exhibited greater weight loss and mortality,elevated disease activity index,as well as higher expression levels of pro-inflammation genes.Thus,Scgn deficiency in mice clearly led to higher DSS sensitivity.Consistent with the in vitro GLP-1 secretion assay,a gut hormone secretion protocol using explanted colon also showed that GLP-1 and GLP-2 secretion in response to DHA and Glucose was severely diminished in Scgn knockout mice.In summary,our studies,combining multiple genetics,cell biology and animal models identified that SCGN is a new genetic risk factor related to pediatric IBD and further elucidated the function and mechanism of SCGN in hormone secretion and intestinal inflammation.The most important insights developed from this study are listed as following:1.We firstly reported that an ultra-rare homozygous nonsynonymous mutant of SCGN,p.R77H,caused early onset ulcerative colitis.2.The mechanism originally illustrated in this study,by which SCGN controls optimal GLP1 secretion process by directly binding to SNAP-25 and regulating its subcellular localization in STC-1 cells.In addition to in vitro discoveries,SCGN also plays a critical role in gut hormone process in vivo.3.SCGN deficient mice had partially reproduced human IBD phenomenon by exhibiting increased susceptibility to DSSinduced colitis.Our discoveries provide new information regarding the genetic etiology of IBD and have filled the knowledge gap about the specific mechanism of SCGN in SNARE complex regulation and hormone release process.