Naturally processed peptides from HLA-DQ7 (alpha1*0501-beta1*0301): influence of both alpha and beta chain polymorphism in the HLA-DQ peptide binding specificity

Self peptides bound to HLA-DQ7 (alpha1*0501-beta1*0301), one of the HLA molecules associated with protection against insulin-dependent diabetes mellitus, were characterized after their acid elution from immunoaffinity-purified HLA-DQ7 (alpha1*0501-beta1*0301) molecules. The majority of these self peptides derived from membrane-associated proteins including HLA class I, class II, class II-associated invariant chain peptide and the transferrin-receptor (TfR). By in vitro binding assays, the specificity of these endogenous peptides for HLA-DQ7 (alpha1*0501-beta1*0301) molecules was confirmed. Among these peptides, the binding specificity of the TfR 215-230 self peptide was further examined on a variety of HLA-DQ and DR dimers. Several findings emerged from this analysis: (1) this peptide displayed HLA-DQ allelic specificity, binding only to HLA-DQ7 (alpha1*0501-beta1*0301); (2) when either the DQalpha or DQbeta chain was exchanged, little or no binding was observed, indicating that specificity of HLA-DQ peptide binding was determined by polymorphic residues of both the alpha and beta chains. (3) Unexpectedly, the TfR 215-230 self peptide, eluted from DQ, was promiscuous with regard to HLA-DR binding. This distinct DR and DQ binding pattern could reflect the structure of these two molecules as recently evidenced by crystallography.     

On the HLA-DQ(alpha 1*0501, beta 1*0201)-associated susceptibility in celiac disease: a possible gene dosage effect of DQB1*0201

The HLA-associated susceptibility to develop celiac disease (CD) seems mainly to be conferred by a particular HLA-DQ heterodimer encoded by the DQA1*0501 and DQB1*0201 genes either in cis or in trans position. To study the possible influence of DRB1 or other DQA1 and DQB1 alleles on the CD susceptibility conferred by these DQ genes, we performed genomic HLA typing of 94 CD patients, selected those who carried at least one copy of the DRB1*0301-DQA1*0501-DQB1*0201 haplotype (N = 89) and compared them to 47 random, healthy Norwegians matched with the patients to carry at least one copy of the above haplotype. We found an excess of DQB1*0201 homozygosity in the patients. This was due to an increased frequency of the DRB1*0301-DQA1*0501-DQB1*0201 and DRB1*0701-DQA1*0201-DQB1*0201 haplotypes present on the other chromosome. We propose that, in individuals carrying the DQA1*0501 and DQB1*0201 alleles, the presence of a second copy of the DQB1*0201 allele increases susceptibility to CD.     

Variation in HLA-DM expression influences conversion of MHC class II alpha beta:class II-associated invariant chain peptide complexes to mature peptide-bound class II alpha beta dimers in a normal B cell line

The maturation of invariant chain (Ii):MHC class II complexes into peptide-loaded alpha beta dimers occurs by proteolytic removal of Ii chain and binding of antigenic peptides derived from exogenous and endogenous Ags. A fragment of the Ii chain (class II-associated invariant chain peptide (CLIP) remains associated with class II alpha beta and is an intermediate in this process. Conversion of alpha beta:CLIP complexes into alpha beta:peptide complexes is facilitated by HLA-DM. Two unique mAbs, specific for I-Ab bound to human CLIP and I-Ab bound to DR alpha peptide, were used to assess the formation of these peptide:class II complexes in a human B lymphoblastoid cell line (B-LCL) (Swei) transfected with I-A(b). In multiple independent Swei:I-Ab transfectants, the amount of human CLIP (hCLIP):I-Ab expressed was inversely proportional to the amount of DR alpha 52-68:I-Ab; quantitative differences in HLA-DM expression accounted for this phenotype. In the low DM transfectant, a substantial proportion of I-Ab, but not DR molecules, was altered structurally and unable to present native protein Ags. Addition of DM transgenes to the DM-low cells resulted in an increase in DR alpha 52-68:I-Ab coupled with a decrease in hCLIP:I-Ab complexes and restoration of exogenous protein Ag presentation. The DR5 molecules in Swei cells, which have a lower affinity for hCLIP than I-Ab, were not affected by low DM expression, suggesting that the amount of DM required for conversion of CLIP:class II to peptide:class II may depend on the affinity of the class II molecules for CLIP or DM.     

A peptide binding motif for HLA-DQA1*0102/DQB1*0602, the class II MHC molecule associated with dominant protection in insulin-dependent diabetes mellitus

The central integration of signals from pulmonary vagal C-fibers (or type-J receptors) with those arising from cardiac, peripheral chemoreceptor, and baroreceptor afferents to neurons within the nucleus of the solitary tract (NTS) was studied in an arterially perfused working heart-brain stem preparation of adult mouse. Pulmonary vagal C-fibers were excited by right atrial injection of phenylbiguanide (PBG) while cardiac receptors were stimulated by left ventricular injection of veratridine (1-3 micrograms/kg) or mechanically by distension of the left ventricle (20-50 microl perfusate) using an indwelling cannula. Carotid body chemoreceptors were activated by aortic injection of Na cyanide, whereas baroreceptors were stimulated by increasing arterial perfusion pressure. Stimulation of pulmonary C-fibers and cardiac, chemo-, and baroreceptors all produced a reflex bradycardia (23-133 bpm). Central respiratory activity, as recorded from the phrenic nerve, was depressed by stimulating pulmonary C-fibers and cardiac and baroreceptors but enhanced in amplitude and frequency during chemoreceptor stimulation. Twenty-seven NTS neurons were excited and three were inhibited after pulmonary C-fiber stimulation displaying decrementing discharges with a peak firing frequency of up to 42 Hz (15 +/- 2.2 Hz, mean +/- SE) that lasted for 8.8 +/- 0.9 s. These responses occurred <1 s from the end of the PBG injection that was within the pulmonary circulation time. None of these cells responded to increases in right atrial pressure. All cells excited by PBG were also driven synaptically after electrical stimulation of the ipsilateral cervical vagus nerve at a latency of 32.9 +/- 3.2 ms (range 20-62 ms). None of these neurons had ongoing activity related to central respiratory activity. Convergence from cardiorespiratory afferents to 21 neurons driven by pulmonary C-fibers was tested. Twenty-five percent of cells were selectively excited by chemical stimulation of cardiac receptors alone, 19% were driven by peripheral chemoreceptors, and 38% responded to both cardiac and chemoreceptor activation. In contrast, only 13% of the cells activated by PBG injection responded to stimulation of baroreceptors and only 6% to cardiac mechanoreceptor stimulation. None of these neurons were activated by increasing right atrial pressure. The data indicate a high proportion of afferent convergence from pulmonary C-fibers, cardiac receptors, and peripheral chemoreceptors in the NTS. However, these neurons appear not to integrate inputs from cardiovascular mechanoreceptors. The significance of the data is discussed in relation to pathological disease states such as pulmonary congestion and cardiac failure.     

Exceptional stability of the HLA-DQA1*0102/DQB1*0602 alpha beta protein dimer, the class II MHC molecule associated with protection from insulin-dependent diabetes mellitus

HLA-DQ alleles are closely associated with susceptibility and resistance to insulin-dependent diabetes mellitus (IDDM) but the immunologic mechanisms involved are not understood. Structural studies of the IDDM-susceptible allele, HLA-DQA1*0301/DQB1*0302, have classified it as a relatively unstable dimer, particularly at neutral pH. This is reminiscent of studies in the nonobese diabetic mouse, in which I-A(g7) is relatively unstable, in contrast to other murine I-A alleles, suggesting a correlation between unstable MHC class II molecules and IDDM susceptibility. We have addressed this question by analysis of dimer stability patterns among various HLA-DQ molecules. In EBV-transformed B-lymphoblastoid cell lines and PBL, the protein encoded by the IDDM-protective allele HLA-DQA1*0102/DQB1*0602 was the most SDS stable when compared with other HLA-DQ molecules, including HLA-DQA1*0102/DQB1*0604, a closely related allele that is not associated with protection from IDDM. Expression of six different HLA-DQ allelic proteins and three different HLA-DR allelic proteins in the bare lymphocyte syndrome cell line, BLS-1, revealed that HLA-DQA1*0102/DQB1*0602 is SDS stable even in the absence of HLA-DM, while other HLA class II molecules are not. These results suggest that the molecular property of HLA-DQ measured by resistance to denaturation of the alphabeta dimer in SDS may play a role in IDDM protection.     

Pocket 4 of the HLA-DR(alpha,beta 1*0401) molecule is a major determinant of T cells recognition of peptide

To investigate the functional roles of individual HLA-DR residues in T cell recognition, transfectants expressing wild-type or mutant DR(alpha,beta 1*0401) molecules with single amino acid substitutions at 14 polymorphic positions of the DR beta 1*0401 chain or 19 positions of the DR alpha chain were used as antigen-presenting cells for five T cell clones specific for the influenza hemagglutinin peptide, HA307-19. Of the six polymorphic positions in the DR beta floor that were examined, mutations at only two positions eliminated T cell recognition: positions 13 (four clones) and 28 (one clone). In contrast, individual mutations at DR beta positions 70, 71, 78, and 86 on the alpha helix eliminated recognition by each of the clones, and mutations at positions 74 and 67 eliminated recognition by four and two clones, respectively. Most of the DR alpha mutations had minimal or no effect on most of the clones, although one clone was very sensitive to changes in the DR alpha chain, with loss of recognition in response to 10 mutants. Mutants that abrogated recognition by all of the clones were assessed for peptide binding, and only the beta 86 mutation drastically decreased peptide binding. Single amino acid substitutions at polymorphic positions in the central part of the DR beta alpha helix disrupted T cell recognition much more frequently than substitutions in the floor, suggesting that DR beta residues on the alpha helix make relatively greater contributions than those in the floor to the ability of the DR(alpha,beta 1*0401) molecule to present HA307-19. The data indicate that DR beta residues 13, 70, 71, 74, and 78, which are located in pocket 4 of the peptide binding site in the crystal structure of the DR1 molecule, exert a major and disproportionate influence on the outcome of T cell recognition, compared with other polymorphic residues.     

Mutation of the alpha 2 domain disulfide bridge of the class I molecule HLA-A*0201. Effect on maturation and peptide presentation

This report documents the introduction of standard-setting in the haemato-oncological reverse-isolation unit of the Kanton hospital, Basle. The topic is the prevention or reduction of nausea, choking and vomiting, and the treatment of patients who are receiving prolonged chemotherapy. The objective was quality assurance in nursing care. The definition of the desired outcome was the well-being and safety of patients. In addition the wish of the staff to increase their security in dealing with antiemetics was taken into consideration. The authors describe the basic problems they encountered in their strategies and actions for standard setting. By extensive use of measurement and evaluation it was possible, in cooperation with medical leadership, to develop instructional leaflets and checklists. Approximation to the intended outcome was achieved. The well being of patients and satisfaction of the safety-needs of staff and patients reached a high level. The report shows that the introduction of nursing standards requires great effort and a high level of commitment of the entire team. It also shows that the effort is worthwhile for patients and staff.     

Specific contribution of Tyk2 JH regions to the binding and the expression of the interferon alpha/beta receptor component IFNAR1

Cytokine signaling involves the activation of the Janus kinase (JAK) family of tyrosine kinases. These enzymes are physically associated with cytokine receptor components. Here, we sought to define the molecular basis of the interaction between Tyk2 and IFNAR1, a component of the interferon alpha/beta receptor, by delimiting a minimal IFNAR1 binding region in the Tyk2 protein. Using an in vitro assay system, we narrowed down the interaction domain to a region comprising the JH7 and part of the JH6 homology boxes (amino acids 22-221). When expressed in Tyk2-negative cells, the JH7-6 region was unable to stabilize IFNAR1 protein levels, a critical function that we previously attributed to the N region (amino acids 1-591) of Tyk2. Moreover, substitution of the JH7-JH6 domain in JAK1 with that of Tyk2 did not restore IFNAR1 level nor interferon alpha signaling in Tyk2-negative cells. Thus, the major interaction surface lies within JH7-6, but additional JH regions (JH5-4-3) contribute in a specific manner to the in vivo assembly of Tyk2 and IFNAR1. Evidence is also provided of the lack of specificity of the Tyk2 kinase-like and tyrosine kinase domains in interferon alpha/beta receptor signaling.     

An alpha 5 beta 1-like integrin receptor mediates the binding of less pathogenic Candida species to fibronectin

The present study was undertaken to investigate whether less pathogenic Candida species (C. tropicalis, C. stellatoidea, C. krusei and C. glabrata) express a fibronectin receptor (FNr) antigenically related to alpha 5 beta 1 integrin, which mediates their binding to fibronectin (FN). By flow cytometric analysis, a monoclonal antibody (MAb) directed against human alpha 5 integrin subunit (clone SAM-1) and two different antisera to FNr positively stained C. tropicalis, C. stellatoidea and C. glabrata, with the greatest expression observed for C. tropicalis. No or only marginal immunoreactivity was found on C. krusei. C. tropicalis, C. stellatoidea, C. glabrata, but not C. krusei yeasts specifically adhered to FN; higher levels of adhesion were found for C. tropicalis and C. stellatoidea with respect to C. glabrata. Less pathogenic Candida spp. bound to the Arg-Gly-Asp (RGD) containing 120-kDa fragment of FN and adhesion to intact FN was markedly inhibited by Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), but not by Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) peptides. In addition, anti-alpha 5 SAM-1 MAb and both anti-FNr antisera strongly blocked binding of less pathogenic Candida spp. to FN. Overall, these results indicate that less pathogenic Candida spp., including C. tropicalis, C. stellatoidea and C. glabrata, express a receptor antigenically related to alpha 5 beta 1 integrin which mediates their adhesion to FN.     

Characterization of the peptide binding motif of a rhesus MHC class I molecule (Mamu-A*01) that binds an immunodominant CTL epitope from simian immunodeficiency virus

The majority of immunogenic CTL epitopes bind to MHC class I molecules with high affinity. However, peptides longer or shorter than the optimal epitope rarely bind with high affinity. Therefore, identification of optimal CTL epitopes from pathogens may ultimately be critical for inducing strong CTL responses and developing epitope-based vaccines. The SIV-infected rhesus macaque is an excellent animal model for HIV infection of humans. Although a number of CTL epitopes have been mapped in SIV-infected rhesus macaques, the optimal epitopes have not been well defined, and their anchor residues are unknown. We have now defined the optimal SIV gag CTL epitope restricted by the rhesus MHC class I molecule Mamu-A*01 and defined a general peptide binding motif for this molecule that is characterized by a dominant position 3 anchor (proline). We used peptide elution and sequencing, peptide binding assays, and bulk and clonal CTL assays to demonstrate that the optimal Mamu-A*01-restricted SIV gag CTL epitope was CTPYDINQM(181-189). Mamu-A*01 is unique in that it is found at a high frequency in rhesus macaques, and all SIV-infected Mamu-A*01-positive rhesus macaques studied to date develop an immunodominant gag-specific CTL response restricted by this molecule. Identification of the optimal SIV gag CTL epitope will be critical for a variety of studies designed to induce CD8+ CTL responses specific for SIV in the rhesus macaque.     

Structure-function relationship of the inhibition of the 3,5,3'-triiodothyronine binding to the alpha1- and beta1-thyroid hormone receptor by amiodarone analogs

Desethylamiodarone (DEA), the major metabolite of the potent antiarrhythmic drug amiodarone (A), acts as a competitive inhibitor of T3, binding to the alpha1-thyroid hormone receptor (alpha1-T3R), but as a noncompetitive inhibitor with respect to the beta1-T3R. To gain insight into the structure- function relationship of the interaction between A metabolites and T3Rs, we investigated the effects of several A analogs on T3 binding to the alpha1-T3R and beta1-T3R in vitro. The analogs tested were: 1) compounds obtained by deethylation of A, DEA, and desdiethylamiodarone (DDEA); 2) compounds obtained by deiodination of A, monoiodoamiodarone and desdiiodoamiodarone (DDIA); and 3) benzofuran derivatives with various iodination grades, 2-butyl-3-(4-hydroxy-3,5-diiodo-benzoyl)benzofuran (L3373, two iodine atoms), L6424 (L3373 with one iodine atom), and L3372 (L3373, no iodine atoms). IC50, values of inhibition of T3 binding to alpha1-T3R and beta1-T3R, respectively, were as follows (mean +/- SD, expressed x 10(-5) M): DEA, 4.7 +/- 0.9 and 2.7 +/- 1.4 (P < 0.001); DDEA, 3.7 +/- 0.9 and 1.9 +/- 0.3 (P < 0.001); monoiodoamiodarone, more than 20 and more than 20; DDIA, 16.2 +/- 5.6 and 9.1 +/- 2.1 (P < 0.01); L3373, 3.8 +/- 1.0 and 3.6 +/- 0.5 (P = NS); L6424, 11.3 +/- 5.7 and 10 +/- 2.0 (P = NS); and L3372, no inhibition. Scatchard analyses in the presence of DDEA, DDIA, and L3373 demonstrated a dose-dependent decrease in Ka, but no change in the maximum binding capacity (MBC) of T3 binding to alpha1-T3R. Langmuir plots clearly indicated competitive inhibition of T3 binding to alpha1-T3R by DDEA, DDIA, and L3373. In contrast, these three analogs acted differently with respect to the beta1-T3R. DDEA and DDIA decreased both Ka and MBC in Scatchard plots using beta1-T3R, demonstrating noncompetitive inhibition. L3373 decreased dose-dependently Ka, but not MBC, values of T3 binding to the beta1-T3R and clearly acted as a competitive inhibitor. Ki plots indicated that DDEA, DDIA, and L3373 do not interfere significantly with occupied T3Rs. KI (inhibition constant for the unoccupied receptor) plots demonstrated increasing inhibition of the T3 binding to unoccupied receptors with increasing analog concentrations. In summary, 1) removal of one or two ethyl groups of A results in compounds with strong but almost equal potency of inhibiting T3R binding, whereas removal of one or two iodine atoms of A has a lower potency in this respect. The strong inhibitory potency of the benzofuran derivative L3373 (equalling that of the deethylated compounds) is lost upon deiodination. 2) All tested A analogs acted as competitive inhibitors to the alpha1-T3R. The behavior to the beta1-T3R was different; deethylation or deiodination of A resulted in noncompetitive inhibition, whereas L3373 was a competitive inhibitor. The potency of deethylated and deiodinated compounds (but not of the benzofuran derivatives) for inhibiting T3 binding was twice as high for the beta1-T3R as for the alpha1-T3R. 3) All tested A analogs preferentially interfere with T3 binding to unoccupied receptors. The implications of these findings for the structure-activity relationship are the following: 1) the size of the diethyl-substituted nitrogen group and of the two bulky iodine atoms in the A molecule hamper the binding of A at the T3 binding site of T3Rs; and 2) differences in the hormone-binding domain of alpha1- and beta1-T3Rs are likely to account for the competitive or noncompetitive nature of inhibition of T3 binding by A analogs.     

A recombinant single-chain human class II MHC molecule (HLA-DR1) as a covalently linked heterotrimer of alpha chain, beta chain, and antigenic peptide, with immunogenicity in vitro and reduced affinity for bacterial superantigens

Major histocompatibility complex (MHC) class II molecules bind to numerous peptides and display these on the cell surface for T cell recognition. In a given immune response, receptors on T cells recognize antigenic peptides that are a minor population of MHC class II-bound peptides. To control which peptides are presented to T cells, it may be desirable to use recombinant MHC molecules with covalently bound antigenic peptides. To study T cell responses to such homogeneous peptide-MHC complexes, we engineered an HLA-DR1 cDNA coding for influenza hemagglutinin, influenza matrix, or HIV p24 gag peptides covalently attached via a peptide spacer to the N terminus of the DR1 beta chain. Co-transfection with DR alpha cDNA into mouse L cells resulted in surface expression of HLA-DR1 molecules that reacted with monoclonal antibodies (mAb) specific for correctly folded HLA-DR epitopes. This suggested that the spacer and peptide did not alter expression or folding of the molecule. We then engineered an additional peptide spacer between the C terminus of a truncated beta chain (without transmembrane or cytoplasmic domains) and the N terminus of full-length DR alpha chain. Transfection of this cDNA into mouse L cells resulted in surface expression of the entire covalently linked heterotrimer of peptide, beta chain, and alpha chain with the expected molecular mass of approximately 66 kDa. These single-chain HLA-DR1 molecules reacted with mAb specific for correctly folded HLA-DR epitopes, and identified one mAb with [MHC + peptide] specificity. Affinity-purified soluble secreted single-chain molecules with truncated alpha chain moved in electrophoresis as compact class II MHC dimers. Cell surface two-chain or single-chain HLA-DR1 molecules with a covalent HA peptide stimulated HLA-DR1-restricted HA-specific T cells. They were immunogenic in vitro for peripheral blood mononuclear cells. The two-chain and single-chain HLA-DR1 molecules with covalent HA peptide had reduced binding for the bacterial superantigens staphylococcal enterotoxin A and B and almost no binding for toxic shock syndrome toxin-1. The unique properties of these engineered HLA-DR1 molecules may facilitate our understanding of the complex nature of antigen recognition and aid in the development of novel vaccines with reduced superantigen binding.     

Disruption of fibronectin binding to the alpha 5 beta 1 integrin stimulates the expression of cyclin-dependent kinases and DNA synthesis through activation of extracellular signal-regulated kinase

The alpha 5 alpha 1 integrin, a fibronectin receptor, has been implicated in the control of cell growth and the regulation of gene expression. We report that disruption of ligation between alpha 5 alpha 1 and fibronectin by integrin alpha 5 subunit or fibronectin monoclonal antibodies stimulated DNA synthesis in growth-arrested FET human colon carcinoma cells. This stimulation only occurred when monoclonal antibody was added in the early G1 phase of the cell cycle after release from quiescence by fresh medium. Stimulation of DNA synthesis by alpha 5 or fibronectin antibody was concentration- and time-dependent. FET cells expressed alpha 4 beta 1 integrin (another fibronectin receptor); however, addition of anti-human integrin alpha 4 monoclonal antibody had no effect on DNA synthesis. Treatment with alpha 5 monoclonal antibody led to a marked increase in the expression of CDK4 in G1 phase of the cell cycle and consequently increased the phosphorylation of retinoblastoma protein. alpha 5 monoclonal antibody treatment increased both cyclin A- and cyclin E-associated kinase activity which was accompanied by increased protein levels of CDK2 and cyclin A. Western blotting of immunoprecipitates demonstrated increased CDK2-cyclin E and CDK2-cyclin A complexes in cells treated with alpha 5 monoclonal antibody. Furthermore, disruption of alpha 5 alpha 1/fibronectin ligation activated mitogen-activated protein kinase p44 and p42 (extracellular signal-regulated kinase 1 and 2). Pretreatment of the cells with a specific inhibitor of MEK-1, PD98059, blocked the alpha 5 monoclonal antibody-induced mitogen-activated protein kinase activity. In addition PD98059 prevented alpha 5 monoclonal antibody-induced DNA synthesis. Since alpha 5 alpha 1 ligation to fibronectin is associated with decreased growth parameters, our results indicate that ligation of alpha 5 alpha 1 integrin to fibronectin results in suppressed mitogen-activated protein kinase activity which in turn inhibits cyclin-dependent kinase activity in growth-arrested cells.     

Human mucosal addressin cell adhesion molecule-1 (MAdCAM-1) demonstrates structural and functional similarities to the alpha 4 beta 7-integrin binding domains of murine MAdCAM-1, but extreme divergence of mucin-like sequences

The mucosal vascular addressin, mucosal addressin cell adhesion molecule-1 (MAdCAM-1), is an Ig family adhesion receptor preferentially expressed by venular endothelial cells at sites of lymphocyte extravasation in murine mucosal lymphoid tissues and lamina propria. MAdCAM-1 specifically binds both human and mouse lymphocytes that express the homing receptor for Peyer's patches, the integrin alpha 4 beta 7. Functional expression cloning was used to isolate a cDNA from a macaque mesenteric lymph node library that encodes the homologue to murine MAdCAM-1. The macaque cDNA was subsequently used to clone the human homologue as well. Expression of human MAdCAM-1 RNA is restricted to mucosal tissues, gut-associated lymphoid tissues and spleen. Human MAdCAM-1 selectively binds both murine and human lymphocyte cell lines expressing alpha 4 beta 7. Human and macaque MAdCAM-1 have two Ig-like domains that are similar to the two amino-terminal integrin binding domains of murine MAdCAM-1. The conservation of sequences within the region homologous to the mucin/IgA domain of murine MAdCAM-1 is, however, much less apparent. These receptors exhibit considerable variation from murine MAdCAM-1 with respect to the length of the mucin-like sequence and the lack of a membrane proximal Ig (IgA-like) domain. The isolation of these different species of MAdCAM-1 demonstrates greater seleoffctive pressure for maintenance of amino acids involved in alpha 4 beta 7 binding than those sequences presumably involved in the presentation of carbohydrates for selectin binding.