ABSTRACT
AIMS/BACKGROUND: The integrin alpha4beta7 and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) are involved in normal recirculation of lymphocytes between the blood and the tissues of the gastrointestinal tract. In this study we have examined the expression of MAdCAM-1 in human liver. METHODS: MAdCAM-1 expression was determined in archival human liver tissues by immunohistochemistry. RESULTS: While MAdCAM-1 was not detected in normal fetal or adult human liver, expression was observed in association with portal tract inflammation in a variety of liver diseases. Detailed analysis of liver biopsies from patients with hepatitis C showed a positive correlation between the portal/periportal component of the histological activity index (HAI) grade and the presence or absence of MAdCAM-1 expression. CONCLUSION: MAdCAM-1 expression may be important in the recruitment of lymphocytes to the liver during inflammation.
Subject(s)
Hepatitis/metabolism , Immunoglobulins/metabolism , Liver/metabolism , Mucoproteins/metabolism , Receptors, Lymphocyte Homing/metabolism , Animals , Antibody Specificity , Antigens, CD34/metabolism , Binding Sites, Antibody , Cell Adhesion Molecules , Enzyme-Linked Immunosorbent Assay , Fetus/metabolism , Hepatitis/pathology , Humans , Immunoenzyme Techniques , Liver/pathology , Rabbits , Receptors, Complement 3d/metabolismABSTRACT
Results from protein mutagenesis and x-ray crystallographic studies of the multidomain protein Vascular Cell Adhesion Molecule (VCAM) were used to design cyclic octapeptides that retain the critical structural and binding elements of the epitope of VCAM in the interaction with the integrin alpha 4 beta 1 (VLA-4). Changes in the activities of peptide analogues correlated with the relative activities of protein mutants of VCAM, and predicted the properties of two new mutants that bound alpha 4 beta 1 with improved affinity vs wild type protein. The nmr structures of two peptides revealed a high degree of similarity to the structure of the VCAM binding epitope. These results demonstrate that a compact binding epitope identified via protein structure-function studies may be transferred to a synthetically accessible small peptide with the key structure-activity relationships intact.
Subject(s)
Epitopes/chemistry , Integrins/metabolism , Peptides, Cyclic/chemistry , Protein Binding , Receptors, Lymphocyte Homing/metabolism , Binding Sites , Binding, Competitive , Integrin alpha4beta1 , Magnetic Resonance Spectroscopy , Methotrexate/chemistry , Models, Molecular , Peptide Fragments/chemistry , Structure-Activity Relationship , Vascular Cell Adhesion Molecule-1/chemistryABSTRACT
The integrin alpha 4 beta 7 and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) are molecules involved in the normal recirculation of lymphocytes between the blood and the gastrointestinal tract. These molecules may play a complementary and significant role in animal models of colitis. We have investigated the structural interaction between alpha 4 beta 7 and MAdCAM-1. Site-directed mutagenesis studies of the MAdCAM-1 molecule has led to the identification of the amino acid residue (LDT) in the loop between beta strands C and D of the Ig-superfamily-like folds being involved in the adhesive and cell activation functions of MAdCAM-1 with alpha 4 beta 7.
Subject(s)
Colitis/immunology , Immunoglobulins , Mucoproteins , Receptors, Lymphocyte Homing , Amino Acid Sequence , Animals , Cell Adhesion Molecules , Colitis/genetics , Humans , Immunoglobulins/chemistry , Immunoglobulins/genetics , Immunoglobulins/immunology , Integrins/chemistry , Integrins/genetics , Integrins/immunology , Molecular Sequence Data , Mucoproteins/chemistry , Mucoproteins/genetics , Mucoproteins/immunology , Mutagenesis, Site-Directed , Receptors, Lymphocyte Homing/chemistry , Receptors, Lymphocyte Homing/genetics , Receptors, Lymphocyte Homing/immunology , Structure-Activity RelationshipABSTRACT
The leukocyte integrin receptor, alpha 4 beta 7, and the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) are postulated to be important in regulating lymphocyte trafficking to normal intestine. Here we provide the first description of MAdCAM-1 expression in inflamed intestine. Using mouse models of experimentally induced colitis, we show a concordant increase in MAdCAM-1 expression associated with increased cellular infiltrates in areas of intestinal inflammation. To understand more of the molecular nature of the interactions between MAdCAM-1 and its leukocyte ligand, the alpha 4 beta 7 integrin receptor, we have analyzed the structural and functional properties of chimeric recombinant MAdCAM-1 proteins in vitro. Using site-directed mutagenesis and molecular modeling, we demarcate the alpha 4 beta 7 binding motif as three linear residues within the C-D loop in the first domain of MAdCAM-1. Mutation of residue L40, D41, or T42 in the first domain completely abrogates alpha 4 beta 7+ cell binding and cellular activation. Mutagenesis of other residues in the first domain do not impact these functions. We have modeled peptides based on the predicted structure of the alpha 4 beta 7 integrin binding motif on MAdCAM-1 and are able to show specific and selective blocking of cell binding. These observations suggest that the amino acid residues LDT on MAdCAM-1 play a role in the interaction with alpha 4 beta 7 in cell adherence and cell activation.
Subject(s)
Immunoglobulins/chemistry , Immunoglobulins/physiology , Integrins/chemistry , Mucoproteins/chemistry , Mucoproteins/physiology , Protein Conformation , Receptors, Lymphocyte Homing/chemistry , Receptors, Lymphocyte Homing/physiology , Amino Acid Sequence , Animals , Binding, Competitive , Cell Adhesion Molecules , Colitis/immunology , Colitis/metabolism , Colitis/pathology , Immunoglobulins/genetics , Integrins/antagonists & inhibitors , Integrins/metabolism , Lymphocyte Activation/drug effects , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Sequence Data , Mucoproteins/genetics , Peptides/pharmacology , Protein Binding/immunology , Recombinant Proteins/immunology , Recombinant Proteins/metabolismABSTRACT
The integrin receptors alpha 4 beta 1 and alpha 4 beta 7 both bind to vascular cell adhesion molecule-1 (VCAM-1). Here, we report that the amino acid residue requirements for murine VCAM-1 adhesion to murine alpha 4 beta 1 (WEHI 231) and alpha 4 beta 7 (38C13/beta 7-transfectant) positive cells are strikingly similar but nonidentical under multiple adhesion activity states. By site-directed mutagenesis of domain 1 of VCAM-1, the amino acid residues on the loop between beta strands C and D (R36, Q38, I39, D40, P42) and on the adjacent antiparallel beta strand F (L70 and T72) were required for basal level adhesion to both alpha 4 beta 1-positive and alpha 4 beta 7-positive cells. Mutation at two other sites, N44 (loop between beta strands C and D) and E66 (loop between beta strands E and F), specifically reduced alpha 4 beta 7-positive cell adhesion, but not alpha 4 beta 1-positive cell adhesion. Mutation H85A augmented alpha 4 beta 7 binding but not alpha 4 beta 1 binding. These apparent differences relate to the higher intrinsic activity state of alpha 4 beta 1 on WEHI 231 than on alpha 4 beta 7 (38C13/beta 7-transfectant). In contrast, under higher adhesion activity states induced by either MnCl2 or truncation of the beta 7 cytoplasmic tail, mutation of either amino acid residue D40 or L70 completely blocked cell adhesion without evidence of structural perturbation of VCAM-1. These results suggested that the two structurally discontinuous amino acid residues, the negatively charged D40 and the hydrophobic L70 adjacently located on domain 1 of VCAM-1, are essential for interaction under multiple activity states with both alpha 4 beta 1 and alpha 4 beta 7 integrin receptors.
Subject(s)
Integrins/metabolism , Receptors, Lymphocyte Homing/metabolism , Vascular Cell Adhesion Molecule-1/metabolism , Amino Acid Sequence , Amino Acids/metabolism , Animals , Antibodies, Monoclonal/immunology , Cell Adhesion , Epitope Mapping , Integrin alpha4beta1 , Manganese/pharmacology , Mice , Mutagenesis, Site-Directed , Tumor Cells, Cultured , Vascular Cell Adhesion Molecule-1/genetics , Vascular Cell Adhesion Molecule-1/immunologyABSTRACT
This study describes the identification of seven amino acid residues of the vascular cell adhesion molecule (VCAM-1) that influence binding to the alpha 4 beta 1 receptor. Using recombinant murine VCAM-1-IgG, which is bound by both mouse (WEHI 231) and human (Ramos) lymphoid cells, two approaches demonstrated the crucial role of the first two NH2-terminal Ig-like domains in binding: (a) blocking monoclonal anti-mouse VCAM-1 antibodies bound to only truncation variants that included the first two domains; (b) site-direct mutagenesis of the first NH2-terminal domain showed that alanine substitution of the amino acid residues R36, D40, K46, S54, N65, T72, and E81 partially or completely reduced adherence by human and/or mouse cells. Of these D40, when mutated to A, N, or K (but not E), showed complete abrogation of adherence by mouse and human cells, as well as inability to bind blocking anti-murine VCAM-1 antibody MVCAM.A429, while not inducing gross structural perturbations in VCAM-1. By molecular modeling, the D40 residue was located on a beta turn connecting two beta strands defined as C and D. The residues R36, K46, S54, N65, T72, and E81, which perturb cell adherence and caused small changes to gross structure, are conformationally near or adjacent to D40. Although these residues, identified as crucial for cell adhesion, are all located in domain 1, it is evident that there is a structural requirement for domains 1 and 2 to be intact so that cell adhesive function can occur.
Subject(s)
Cell Adhesion Molecules/metabolism , Cell Adhesion/physiology , Integrins/metabolism , Receptors, Antigen, B-Cell/metabolism , Receptors, Very Late Antigen/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal , Base Sequence , Cell Adhesion/genetics , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/immunology , Cloning, Molecular , DNA Mutational Analysis , Epitopes , Humans , Immunoglobulin G/genetics , Integrin alpha4beta1 , Lymphocytes/metabolism , Mice , Models, Molecular , Molecular Sequence Data , Protein Binding , Receptors, Antigen, B-Cell/genetics , Receptors, Antigen, B-Cell/immunology , Receptors, Very Late Antigen/immunology , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Structure-Activity Relationship , Vascular Cell Adhesion Molecule-1Subject(s)
Gene Expression , Protein Sorting Signals/genetics , Proteins/genetics , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Cloning, Molecular/methods , Glycosylation , Molecular Sequence Data , Plasmids , Protein Biosynthesis , Protein Processing, Post-Translational , RNA, Messenger/genetics , Recombinant Fusion Proteins/biosynthesisABSTRACT
Poliovirus type 1 cDNA was prepared from viral RNA encoding the VP1 capsid region of the virus by using a specific DNA primer and was cloned in Escherichia coli. DNA fragments corresponding to VP1 amino acid positions 129 to 302 (pPM5k3), 52 to 302 (pPMhae3), and 24 to 129 (pPMDxba) were incorporated into plasmid vectors designed to express Trp LE-poliovirus VP1 fusion proteins under the control of the inducible tryptophan promoter-operator system. Induction of bacterial cultures containing the plasmids resulted in the production of fusion proteins which accounted for 21% (pPMhae3), 68% (pPM5k3), and 27% (pPMDxba) of the total cell protein. The proteins were purified, and each reacted with polyclonal antibodies raised against intact virions as measured by an enzyme-linked immunosorbent assay. The sera from rabbits immunized with the bacterially produced fusion proteins pPMDxba and pPMhae3 contained poliovirus-neutralizing antibodies.
Subject(s)
Capsid/immunology , Poliovirus Vaccine, Inactivated/genetics , Poliovirus/immunology , Recombinant Fusion Proteins/immunology , Recombinant Proteins/immunology , Animals , Capsid/genetics , Capsid Proteins , Cloning, Molecular , DNA/genetics , Escherichia coli , Molecular Weight , Neutralization Tests , Plasmids , Poliovirus Vaccine, Inactivated/immunology , RabbitsABSTRACT
Mammalian cell lines have been engineered to produce a secreted form of the AIDS retrovirus envelope glycoprotein. The recombinant protein has been isolated from growth-conditioned culture media and used to immunize animals. Antibodies directed against the recombinant molecule were found to react with the envelope glycoprotein produced in virus-infected cells. Furthermore, these antibodies were able to directly inactivate the AIDS retrovirus in a neutralization assay in vitro. The expression system reported here should provide sufficient quantities of the AIDS retrovirus envelope protein for biological and vaccination studies.