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Identification of the amino acids responsible for cholecystokinin receptor subtype selectivity for gastrin
April 1 9 9 5
• MUCIN GENES AS M A R K E R S FOR T H E GENETIC PREDISPOSITION OF ULCERATIVE COLITIS. I.E. Pladdet, A.S. Peda, C.B:M. Oudejans. Department of gastroenterology and Central chemical laboratory, Free University Hospital, Amsterdam, The Netherlands, Mucoglycoproteins pla~ an important role in the protection of the mucosa of the gastrointestinal tract. It is known from the literature that there are changes in mucin contents in patients with Ulcerative Colitis (UC) and a selective depletion of colonic mu¢in subclass during discontinuous salt gradient in UC has been shown. This ~iltered glyeoprotein composition has been found in identical twins with ulcerative colitis, suggesting a genetically defined defect. The. genes which Cod¢~for gastrointestinal mucus have been recently identified. Different alleles can be defined by tandem repeats of variable length using restriction digestion of DNA with appropriate enzymes and southern blotting with chemiluminiscent labelled probes. Methods: We studied MUC 2 and MUC 3 genes in 18 patients with ulcerative colitis, 11 patients with coeliac disease, and 15 patients with diabetes mellitus, Genomic DNA was extracted by a high salt method and digested to completion by restriction enzymes. Gel electrophoreses and Southern blotting Was performed; and afterwards Southe~ hybridization with probe SIB139 and 8Muc40 was performed. Chemiluminescence with anti-digoxigenin conjugated with alkaline phosphatase was performed to identify specific patterns of VNTR's in the different patients groups, Results: We identified four patterns with bands tit~the 9.4 kb, 9.4 kb and 7.9 kb, 7.9 kb, or 7.9 kb and 7.0 kb region for the SMUC40 probe which is specific for the MUC2 gene. Using the SIB139 probe which is specific for the MUC3 gene we only identified one band in the 23.1 kb region in all patients. Patients with UC showed significant more often a pattern of bands in the 9.4 and 7.9 kb region than in the other patient groups (p=0.0163: Fisher exact test). We found in this pilot study a difference in the VNTR pattern of MUC2 gene in patients with UC compared with controls. This finding suggest that MUC2 gene may be a marker in the genetic predisposition of ulcerative colitis. In this case mucin content of the colon in patients with ulcerative colitis is not due to the disease but a predisposing abnormality. Further studies are indicated.
• IDENTIFICATION OF THE AMINO ACIDS RESPONSIBLE FOR CHOLECYSTOKININ RECEPTOR SUBTYPE SELECTIVITY FOR GASTRIN S.S. Poirot and S.A. Wank,. Digestive Diseases Branch, NIDDK, National Institutes of Health, Betbesda, MD 20892 The structurally related receptor subtypes for CCK, CCKAR and CCKBR, are seven transmembrane (TM) G protein-coupled receptors having = 50% amino acid homology. CCKARs are found predominately in the gastrointestinal tract mediating gallbladder contraction and pancreatic enzyme secretion and in localized areas of the nervous system regulating satiety. CCKBRs are also present in the gastrointestinal tract mediating acid secretion and motility and throughout the nervous system regulating anxiety, dopamine dependent behavior, and response to analgesia. CCKARs and CCKBRs can be pharmacologically distinguished on the basis of their affinity for CCK and gastrin, two structurally and functionally related families of peptide agonists with identical COOH-terminal pentapeptide sequences and differing sulfation at the sixth (gastrin)i and seventh (CCK) tyrosyl residues as well as two nonpeptide, substituted benz0diazepine antagonists, L-364,718 (CCKAR selective) and L-365,260 (CCKBR selective). Both CCKARs and CCKBRs have nearly equal affinity for sulfated CCK-8, while CCKBRs have a 500-1000-fold higher affinity for gastrin compared to CCKARs. To determine the structural basis for CCK receptor subtype selectivity for agonists, we synthesized cDNA sequences encoding for chimeric rat CCKARs and CCKBRs and determined their affinity for CCK and gastrin. Restriction endonuclease sites shared either naturally or by site specific mutagenesis between CCKARs and CCKBRs at five locations between TM I and TM VII were chosen to create a CCKBR replaced either progressively or in segments by CCKAR sequence. These chimeric CCKA/B cDNA sequences were transiently expressed in COS-1 cells using a restriction endonuelease modified version of the mammalian expression vector, pCDLSRet, and assayed for their affinity for 1251-BH-CCK-8 alone and in the presence of the agonists CCK-8 and gastrin-17-I and the antagonists L-364,718 and L-365,260, The insertion of restriction endonuclease sites, some of which altered the amino acid sequence of the receptors, had no effect on their ligand affinity compared to wild type receptor. The minimal CCKAR sequence; allowed by the location of the restriction endonuclease sites, that was capable of creating a CCKAIB chimeric receptor with CCKAR subtype selectivity for agonists spanned the region from TM III to TM V, This chimeric CCKA/B receptor retained full affinity for CCK-8 but, unlike the wild type CCKBR, lost its affinity for gasttin-17-I by a factor of 500 to 1000-fold. Interestingly, the affinity for L-364,718 did not change while the affinity for L-365.260 decreased approximately 50-fold compared to wild type CCKBR. Using site directed mutagenic strategies, we have subsequently identified a maximun of 5 amino acids within this area responsible for this change in selectivity for gasttin-17-l. These results present for the first time the amino acid sequence that can completely account for CCK receptor subtype specifity.
Immunology, Microbiology, and Inflammatory Disorders
A895
THE TUMOR NECROSIS FACTOR (TNF) MICROSATELLITE HAPLOTYPE A2B1C2D4E1 CORRELATES WITH INCREASED TNF PRODUCTION IN CROHN'S DISEASE (CD). ~._E_Plevy, H Yang, NM Carramanzana, D Fernandez, J Gaiennie, JI Rotter, H Toyoda, SR Targan. Inflammatory Bowel Disease Center, Cedars-Sinai Research Institute, Los Angeles, CA, 90048. We previously reported a CD associated TNF microsatetlite haplotype, TNFa2blc2d4el, present in 24% of CD patients, 4% of ulcerative colitis (UC) patients, and 7% of ethnically matched controls. Recent data showing efficacy of chimeric anti-TNF-a rnonoclonal antibodies in CD support an important role for this molecule in intestinal inflammation. Heterogeneity in TNF production has been described in IBD and may be determined by genetic heterogeneity at the TNF locus. Therefore, we studied 27 CD patients and 20 UC patients to correlate the TNF microsatellite genotype at 5 loci with TNF production from peripheral blood mononuclear cells (PBMC). All patients were Caucasian. All studies were performed blinded. TNF mierosatellite analysis of 5 markers (TNFa,b,c,d,e) was performed on genomic DNA by polymerase chain reaction. PBMC were isolated from the same patients and 2x106 cells were activated with ConA at 10 p.g/ml and PMA at 2 ng/ml for 24 hours. Total TNF activity was determined by L929 cytotoxicity assay. TNF levels were log transformed for statistical analyses. Higher TNF l~roduction was seen in PBMC from the CD group (n=27), 2366 pg/2xl0 o cells, compared to the UC group (n=20), 262 pg/2xl06 cells (p=0.02). CD patients were then stratified based on the presence of the TNFa2blc2d4el haplotype: CD patients who had the haplotype (n=7) had higher TNF production, 4285 pg/2xl06 cells, than patients who did not have the haplotype (n=20), 1923 pg/2xl06 cells, although this comparison does not reach statistical significance (p=0.1). Since it is not known which, if any, individual allele that comprises the CD associated haplotype may correlate with differences in TNF production, we then compared TNF production in CD patients with TNFa2blc2d4el (n=7), 4285 pg/2xl06 ceils, with CD patients who did not have any alleles contained in the haplotype (n=9), 1227 pg/2xl06 cells. This difference is statistically significant (p=0.04), When CD patients with any of the TNFa2blc2d4el alleles are excluded from analysis, there was no difference in TNF production between the remaining CD patients and UC patients (p=0.7). Conclusions: 1) TNF production from PBMC is higher in CD patients than UC patients. 2) There is heterogeneity in TNF production among CD patients which appears to be due to genetic differences at the TNF locus. 3) The subgroup of CD patients having the TNF microsatellite haplotype a 2 b l c 2 d 4 e l appears to account for the increased TNF production in the CD population. Supported by the Glaxo Institute for Digestive Health, and NIH grant DK43211.
• CROHN'S DISEASE: EVIDENCE FOR GENETIC HETEROGENEITY AND "ANTICIPATION". Polito J, Tokayer A, Childs B, Harris M, Bayless T, The Johns Hopkins Hospital, Baltimore, MD Evidence for the existence of clinical subtypes of Crohn's disease supports the concept of genetic heterogeneity. Genetic "anticipation" refers to increased severity and earlier onset of disease in subsequent generations of affected families. Identification of nucleotide triplet repeats in certain monogenic disorders provides a possible biologic basis for this concept, This study examines a hypothesis of genetic influences on age at diagnosis, phenotypic subtypes and severity of disease, as well as the possible existence of genetic "anticipation" in patients with a family history of Crohn's disease as compared to those without. Methods: Records of 555 consecutive patients with Crohn's disease (CD) were retrospectively reviewed. The age at diagnosis, family history of CD, distribution and subtype of disease, and surgical and complication history were analyzed. Affected family members of patients with CD were similarly compared. Results: Younger age at diagnosis (age < 20 yrs) was associated with a higher prevalence of a positive family history of CD (30% vs 13.4%, p<.005), greater small bowel involvement (88.7% vs 56.7%, p<.O005L stricturing disease (45.8% vs 28.4%, p < .005), and a higher prevalence of surgery for CD (70.6% vs 55.2%, p <.001 ). Older age at diagnosis (> = 4 0 vs < 20yrs) was associated with a higher prevalence of colonic disease (85% vs 71.2%, p< .05) and the inflammatory subtype (55.2% vs 34.5%, p < .0005). The 96 of 555 patients (17%) who had a family history of CD had an earlier age at diagnosis (21.8 vs 26.7 yrs, p<.005) than did those without a family history and a greater prevalence of surgery for abscess or perforation (26% VS 17%, p<.05). There were 27 pairs of 2 generation 1st degree'relatives. Members of the succeeding generation had a younger age at diagnosis (18.93 vs 31.3Syrs, p < .0001 ) than did the parents. In only 5 of the 27 pairs was CD less extensive in the children than in the parents. More extensive disease in the child was more frequently transmitted by male parents ~13 of 16 pairs). Conclusions: Early age at diagnosis appears to define phenotypes of CD patients with more severe disease and a greater likelihood of having similarly affected relatives. Parent-child transmission of CD is associated with earlier onset and more extensive disease, fitting with a concept of genetic "anticipation" for CD.
• MUCIN GENES AS M A R K E R S FOR T H E GENETIC PREDISPOSITION OF ULCERATIVE COLITIS. I.E. Pladdet, A.S. Peda, C.B:M. Oudejans. Department of gastroenterology and Central chemical laboratory, Free University Hospital, Amsterdam, The Netherlands, Mucoglycoproteins pla~ an important role in the protection of the mucosa of the gastrointestinal tract. It is known from the literature that there are changes in mucin contents in patients with Ulcerative Colitis (UC) and a selective depletion of colonic mu¢in subclass during discontinuous salt gradient in UC has been shown. This ~iltered glyeoprotein composition has been found in identical twins with ulcerative colitis, suggesting a genetically defined defect. The. genes which Cod¢~for gastrointestinal mucus have been recently identified. Different alleles can be defined by tandem repeats of variable length using restriction digestion of DNA with appropriate enzymes and southern blotting with chemiluminiscent labelled probes. Methods: We studied MUC 2 and MUC 3 genes in 18 patients with ulcerative colitis, 11 patients with coeliac disease, and 15 patients with diabetes mellitus, Genomic DNA was extracted by a high salt method and digested to completion by restriction enzymes. Gel electrophoreses and Southern blotting Was performed; and afterwards Southe~ hybridization with probe SIB139 and 8Muc40 was performed. Chemiluminescence with anti-digoxigenin conjugated with alkaline phosphatase was performed to identify specific patterns of VNTR's in the different patients groups, Results: We identified four patterns with bands tit~the 9.4 kb, 9.4 kb and 7.9 kb, 7.9 kb, or 7.9 kb and 7.0 kb region for the SMUC40 probe which is specific for the MUC2 gene. Using the SIB139 probe which is specific for the MUC3 gene we only identified one band in the 23.1 kb region in all patients. Patients with UC showed significant more often a pattern of bands in the 9.4 and 7.9 kb region than in the other patient groups (p=0.0163: Fisher exact test). We found in this pilot study a difference in the VNTR pattern of MUC2 gene in patients with UC compared with controls. This finding suggest that MUC2 gene may be a marker in the genetic predisposition of ulcerative colitis. In this case mucin content of the colon in patients with ulcerative colitis is not due to the disease but a predisposing abnormality. Further studies are indicated.
• IDENTIFICATION OF THE AMINO ACIDS RESPONSIBLE FOR CHOLECYSTOKININ RECEPTOR SUBTYPE SELECTIVITY FOR GASTRIN S.S. Poirot and S.A. Wank,. Digestive Diseases Branch, NIDDK, National Institutes of Health, Betbesda, MD 20892 The structurally related receptor subtypes for CCK, CCKAR and CCKBR, are seven transmembrane (TM) G protein-coupled receptors having = 50% amino acid homology. CCKARs are found predominately in the gastrointestinal tract mediating gallbladder contraction and pancreatic enzyme secretion and in localized areas of the nervous system regulating satiety. CCKBRs are also present in the gastrointestinal tract mediating acid secretion and motility and throughout the nervous system regulating anxiety, dopamine dependent behavior, and response to analgesia. CCKARs and CCKBRs can be pharmacologically distinguished on the basis of their affinity for CCK and gastrin, two structurally and functionally related families of peptide agonists with identical COOH-terminal pentapeptide sequences and differing sulfation at the sixth (gastrin)i and seventh (CCK) tyrosyl residues as well as two nonpeptide, substituted benz0diazepine antagonists, L-364,718 (CCKAR selective) and L-365,260 (CCKBR selective). Both CCKARs and CCKBRs have nearly equal affinity for sulfated CCK-8, while CCKBRs have a 500-1000-fold higher affinity for gastrin compared to CCKARs. To determine the structural basis for CCK receptor subtype selectivity for agonists, we synthesized cDNA sequences encoding for chimeric rat CCKARs and CCKBRs and determined their affinity for CCK and gastrin. Restriction endonuclease sites shared either naturally or by site specific mutagenesis between CCKARs and CCKBRs at five locations between TM I and TM VII were chosen to create a CCKBR replaced either progressively or in segments by CCKAR sequence. These chimeric CCKA/B cDNA sequences were transiently expressed in COS-1 cells using a restriction endonuelease modified version of the mammalian expression vector, pCDLSRet, and assayed for their affinity for 1251-BH-CCK-8 alone and in the presence of the agonists CCK-8 and gastrin-17-I and the antagonists L-364,718 and L-365,260, The insertion of restriction endonuclease sites, some of which altered the amino acid sequence of the receptors, had no effect on their ligand affinity compared to wild type receptor. The minimal CCKAR sequence; allowed by the location of the restriction endonuclease sites, that was capable of creating a CCKAIB chimeric receptor with CCKAR subtype selectivity for agonists spanned the region from TM III to TM V, This chimeric CCKA/B receptor retained full affinity for CCK-8 but, unlike the wild type CCKBR, lost its affinity for gasttin-17-I by a factor of 500 to 1000-fold. Interestingly, the affinity for L-364,718 did not change while the affinity for L-365.260 decreased approximately 50-fold compared to wild type CCKBR. Using site directed mutagenic strategies, we have subsequently identified a maximun of 5 amino acids within this area responsible for this change in selectivity for gasttin-17-l. These results present for the first time the amino acid sequence that can completely account for CCK receptor subtype specifity.
Immunology, Microbiology, and Inflammatory Disorders
A895
THE TUMOR NECROSIS FACTOR (TNF) MICROSATELLITE HAPLOTYPE A2B1C2D4E1 CORRELATES WITH INCREASED TNF PRODUCTION IN CROHN'S DISEASE (CD). ~._E_Plevy, H Yang, NM Carramanzana, D Fernandez, J Gaiennie, JI Rotter, H Toyoda, SR Targan. Inflammatory Bowel Disease Center, Cedars-Sinai Research Institute, Los Angeles, CA, 90048. We previously reported a CD associated TNF microsatetlite haplotype, TNFa2blc2d4el, present in 24% of CD patients, 4% of ulcerative colitis (UC) patients, and 7% of ethnically matched controls. Recent data showing efficacy of chimeric anti-TNF-a rnonoclonal antibodies in CD support an important role for this molecule in intestinal inflammation. Heterogeneity in TNF production has been described in IBD and may be determined by genetic heterogeneity at the TNF locus. Therefore, we studied 27 CD patients and 20 UC patients to correlate the TNF microsatellite genotype at 5 loci with TNF production from peripheral blood mononuclear cells (PBMC). All patients were Caucasian. All studies were performed blinded. TNF mierosatellite analysis of 5 markers (TNFa,b,c,d,e) was performed on genomic DNA by polymerase chain reaction. PBMC were isolated from the same patients and 2x106 cells were activated with ConA at 10 p.g/ml and PMA at 2 ng/ml for 24 hours. Total TNF activity was determined by L929 cytotoxicity assay. TNF levels were log transformed for statistical analyses. Higher TNF l~roduction was seen in PBMC from the CD group (n=27), 2366 pg/2xl0 o cells, compared to the UC group (n=20), 262 pg/2xl06 cells (p=0.02). CD patients were then stratified based on the presence of the TNFa2blc2d4el haplotype: CD patients who had the haplotype (n=7) had higher TNF production, 4285 pg/2xl06 cells, than patients who did not have the haplotype (n=20), 1923 pg/2xl06 cells, although this comparison does not reach statistical significance (p=0.1). Since it is not known which, if any, individual allele that comprises the CD associated haplotype may correlate with differences in TNF production, we then compared TNF production in CD patients with TNFa2blc2d4el (n=7), 4285 pg/2xl06 ceils, with CD patients who did not have any alleles contained in the haplotype (n=9), 1227 pg/2xl06 cells. This difference is statistically significant (p=0.04), When CD patients with any of the TNFa2blc2d4el alleles are excluded from analysis, there was no difference in TNF production between the remaining CD patients and UC patients (p=0.7). Conclusions: 1) TNF production from PBMC is higher in CD patients than UC patients. 2) There is heterogeneity in TNF production among CD patients which appears to be due to genetic differences at the TNF locus. 3) The subgroup of CD patients having the TNF microsatellite haplotype a 2 b l c 2 d 4 e l appears to account for the increased TNF production in the CD population. Supported by the Glaxo Institute for Digestive Health, and NIH grant DK43211.
• CROHN'S DISEASE: EVIDENCE FOR GENETIC HETEROGENEITY AND "ANTICIPATION". Polito J, Tokayer A, Childs B, Harris M, Bayless T, The Johns Hopkins Hospital, Baltimore, MD Evidence for the existence of clinical subtypes of Crohn's disease supports the concept of genetic heterogeneity. Genetic "anticipation" refers to increased severity and earlier onset of disease in subsequent generations of affected families. Identification of nucleotide triplet repeats in certain monogenic disorders provides a possible biologic basis for this concept, This study examines a hypothesis of genetic influences on age at diagnosis, phenotypic subtypes and severity of disease, as well as the possible existence of genetic "anticipation" in patients with a family history of Crohn's disease as compared to those without. Methods: Records of 555 consecutive patients with Crohn's disease (CD) were retrospectively reviewed. The age at diagnosis, family history of CD, distribution and subtype of disease, and surgical and complication history were analyzed. Affected family members of patients with CD were similarly compared. Results: Younger age at diagnosis (age < 20 yrs) was associated with a higher prevalence of a positive family history of CD (30% vs 13.4%, p<.005), greater small bowel involvement (88.7% vs 56.7%, p<.O005L stricturing disease (45.8% vs 28.4%, p < .005), and a higher prevalence of surgery for CD (70.6% vs 55.2%, p <.001 ). Older age at diagnosis (> = 4 0 vs < 20yrs) was associated with a higher prevalence of colonic disease (85% vs 71.2%, p< .05) and the inflammatory subtype (55.2% vs 34.5%, p < .0005). The 96 of 555 patients (17%) who had a family history of CD had an earlier age at diagnosis (21.8 vs 26.7 yrs, p<.005) than did those without a family history and a greater prevalence of surgery for abscess or perforation (26% VS 17%, p<.05). There were 27 pairs of 2 generation 1st degree'relatives. Members of the succeeding generation had a younger age at diagnosis (18.93 vs 31.3Syrs, p < .0001 ) than did the parents. In only 5 of the 27 pairs was CD less extensive in the children than in the parents. More extensive disease in the child was more frequently transmitted by male parents ~13 of 16 pairs). Conclusions: Early age at diagnosis appears to define phenotypes of CD patients with more severe disease and a greater likelihood of having similarly affected relatives. Parent-child transmission of CD is associated with earlier onset and more extensive disease, fitting with a concept of genetic "anticipation" for CD.