Human intelectin-1 (ITLN1) genetic variation and intestinal expression

Abstract

Intelectins are ancient carbohydrate binding proteins, spanning chordate evolution and implicated in multiple human diseases. Previous GWAS have linked SNPs in ITLN1 (also known as omentin) with susceptibility to Crohn's disease (CD); however, analysis of possible functional significance of SNPs at this locus is lacking. Using the Ensembl database, pairwise linkage disequilibrium (LD) analyses indicated that several disease-associated SNPs at the ITLN1 locus, including SNPs in CD244 and Ly9, were in LD. The alleles comprising the risk haplotype are the major alleles in European (67%), but minor alleles in African superpopulations. Neither ITLN1 mRNA nor protein abundance in intestinal tissue, which we confirm as goblet-cell derived, was altered in the CD samples overall nor when samples were analyzed according to genotype. Moreover, the missense variant V109D does not influence ITLN1 glycan binding to the glycan β-D-galactofuranose or protein-protein oligomerization. Taken together, our data are an important step in defining the role(s) of the CD-risk haplotype by determining that risk is unlikely to be due to changes in ITLN1 carbohydrate recognition, protein oligomerization, or expression levels in intestinal mucosa. Our findings suggest that the relationship between the genomic data and disease arises from changes in CD244 or Ly9 biology, differences in ITLN1 expression in other tissues, or an alteration in ITLN1 interaction with other proteins.

Figure 1

ITLN1 polymorphisms. (a) The physical arrangement of ITLN1 relative to centromere/telomere orientation and flanking genes on chromosome 1q23.3 is diagramed, along with single nucleotide polymorphisms associated with human disease, including IBD: rs4656940, rs11265501, rs12058717, rs11578770, rs4656953 , rs1333062, rs2274907, rs2274908, rs2274910, rs2297559, and rs4656958^–. Red text highlights GWAS-identified SNPs. (b) 1000 Genomes Project Phase 3 super population allele frequency (percentages) of rs2274907 (A/T, V109D): AFR, African; AMR, American, EAS, East Asian; EUR, European; SAS, South Asian^,. (c) 3D structure of human trimeric ITLN1 bound to β-D-galactofuranose (black). The distance between V109 residue (red) and coordinating calcium (green) are indicated on each monomer (PDB: 4WMY). (d) Amino acid sequence adjacent to coding variant rs2274907 (V109D) in orthologs of human ITLN1 (“—” identical residue present ITLN1).

Figure 2

ITLN1 mRNA and protein expression in the small intestine and colon. (a) ITLN1 mRNA expression in surgical specimens of ileum and colon according to diagnosis. The mRNA copy number per 10 ng total RNA was determined with quantitative real-time RT-PCR using external cDNA standards (see methods). (b) ITLN1 mRNA expression by V109D genotype (excluding ulcerative colitis samples). Box plots present the median and first and third quartiles, whiskers present the range. Statistical analysis of ITLN1 mRNA expression was performed using either a Kruskal–Wallis test with Dunn’s multiple comparisons or two-tailed Mann–Whitney U test; * p < 0.05. (c) Representative real-time bio-layer interferometry responses of recombinant ITLN1 V109 and D109 binding to immobilized β-D-galactofuranose (upper). Responses at 490 s were plotted against ITLN1concentration and fit to a single site binding equation to determine Kd (lower). Data is representative of three separate experiments. (d) SDS-PAGE (4–20% gradient) immunoblots of human ileum by V109D genotype (left, n = 2 per genotype) and disease state (right). (e) Immunohistochemistry of human colon using the following antibodies: ITLN conserved (ITLN^CONS), ITLN1-specific (ITLN1^NTERM), MUC2 mature (MUC2^C3), deglycosylated MUC2 (MUC2^APO, PH1900). Tissue counterstained by DAPI (blue). (f) Immunofluorescence of human colon using ITLN^CONS-FLOR (anti-chicken; Alexa Flour 647) and MUC2^C3 (anti-rabbit; Alexa Flour 488). (g) Immunohistochemistry of human ileum; ITLN2-specific (ITLN2^NTERM) antibody. Red box indicates Paneth cells identified with the ITLN^CONS antibody. CTRL = control (non-Crohn’s disease and non-ulcerative colitis), CD = Crohn’s disease, UC = ulcerative colitis. Images in (e–g) are representative of multiple (> 6 fields) from 9 specimens. Light microscopy scale bars: 20x (200 µm), 40x (100 µm), and 100x (50 µm); fluorescence microscopy scale bars: 20x (100 µm) and 63x (50 µm).