Sialic acid cyclization of human Th homing receptor glycan associated with recurrent exacerbations of atopic dermatitis.

BACKGROUND: The molecular pathogenesis underlying recurrent exacerbations of atopic dermatitis (AD) is unclear. Some peripheral CCR4(+) and CCR7(+) helper memory T cells express the specific homing receptor, sialyl 6-sulfo Lewis X (G152 glycan). This glycan loses receptor activity via cyclization of its sialic acid moiety, thus becoming cyclic sialyl 6-sulfo Lewis X (G159 glycan). These findings suggest that the disordered expression of G152 and G159 glycans may be associated with recurrent exacerbations of AD. OBJECTIVE: To assess the possible association of G152 and G159 glycans, which are expressed on peripheral helper T (Th) cells, with frequency of exacerbations. METHODS: The percentage of glycan-expressing cells among peripheral blood CD4(+)CD45RO(+) lymphocytes was determined by flow cytometry. The association of glycans with the frequency of exacerbations determined by recurrence scores as well as with current disease activity was statistically tested. RESULTS: Current disease activity was significantly associated with CCR4(+)CCR7(-) memory Th cells expressing CSLEX-1 glycan, the conventional skin-trafficking receptor without sialic-acid-cyclization activity. In contrast, the frequency of exacerbations was positively and negatively associated with CCR4(+)CCR7(+) memory Th cells expressing G152 and G159 glycans, respectively. Receiver operating characteristics analyses indicated that the ratio of the G152(+)/G159(+) cell percentages discriminated patients with highly recurrent AD with the best accuracy. CONCLUSION: Flow cytometric determination of G159 and G152 glycans on peripheral helper memory T cells may be clinically useful for identifying patients with highly recurrent AD. Disordered sialic acid cyclization of G152 glycan may underlie highly recurrent AD, which may provide a novel therapeutic approach.

A novel GGA-binding site is required for intracellular sorting mediated by stabilin-1.

Stabilin-1 is a unique scavenger receptor that combines endocytic and intracellular sorting functions in macrophages. Stabilin-1 mediates the endocytosis of acetylated low-density lipoprotein (acLDL), SPARC, and growth hormone family member placental lactogen (PL). At the same time, stabilin-1 is involved in trans-Golgi network-to-endosome routing of the endogenous chitinase-like protein SI-CLP (stabilin-interacting chitinase-like protein). A DDSLL motif in the cytoplasmic tail of stabilin-1 interacts with GGA adaptors; however, the deletion of DDSLL reduces but does not abrogate this interaction. Here, we identified a novel GGA-binding site, EDDADDD, in the cytoplasmic tail of stabilin-1. The deletion of EDDADDD impaired and the deletion of both the DDSLL and EDDADDD sites abrogated the interaction of stabilin-1 with GGAs. The surface exposure of stabilin-1 and stabilin-1-mediated endocytosis of acLDL, SPARC, and PL were not affected by the deletion either of DDSLL or EDDADDD or both. At the same time, both GGA-binding sites were necessary for the intracellular sorting of SI-CLP performed by stabilin-1. Our data indicate that the novel GGA-binding site EDDADDD is essential for stabilin-1-mediated intracellular sorting but is not required for endocytosis.

Stabilin-1 mediates phosphatidylserine-dependent clearance of cell corpses in alternatively activated macrophages.

Stabilin-1 is specifically expressed in alternatively activated macrophages. These macrophages participate in anti-inflammatory and healing processes, and display a high phagocytic capacity. In this study, we provide evidence that stabilin-1 is a membrane receptor that performs a crucial function in the clearance of cell corpses. Stabilin-1 is expressed on the cell surface of alternatively activated macrophages and is recruited to the sites of recognition and engulfment of apoptotic bodies, as well as to early phagosomes. Blocking stabilin-1 in macrophages results in defective engulfment of aged red blood cells. Ectopic expression of stabilin-1 induces the binding and engulfment of aged cells in mouse fibroblast L cells. The binding and phagocytosis are dependent on phosphatidylserine (PS), which is well known as an engulfing ligand. Furthermore, using PS-coated beads, we demonstrate that PS directly interacts with stabilin-1 and is sufficient for stabilin-1-mediated phagocytosis. EGF-like domain repeat in stabilin-1 is responsible for PS recognition and binding. Thus, our results demonstrate that stabilin-1, found on alternatively activated macrophages, is a phagocytic receptor mediating the clearance of apoptotic cells in a PS-dependent manner. Therefore, this protein might play an important role in the maintenance of tissue homeostasis and prevention of autoimmunity.

Rapid plasma membrane-endosomal trafficking of the lymph node sinus and high endothelial venule scavenger receptor/homing receptor stabilin-1 (FEEL-1/CLEVER-1).

The sinusoidal endothelia of liver, spleen, and lymph node are major sites for uptake and recycling of waste macromolecules through promiscuous binding to a disparate family of scavenger receptors. Among the most complex is stabilin-1, a large multidomain protein containing tandem fasciclin domains, epidermal growth factor-like repeats, and a C-type lectin-like hyaluronan-binding Link module, which functions as an endocytic receptor for acetylated low density lipoprotein and advanced glycation end products. Intriguingly, stabilin-1 has also been reported to mediate both homing of leukocytes across lymph node high endothelial venules and adhesion of metastatic tumor cells to peritumoral lymphatic vessels. Currently, however, it is not clear how stabilin-1 mediates these distinct functions. To address the issue, we have investigated the tissue and subcellular localization of stabilin-1 in detail and assessed the functional status of its Link module. We show that stabilin-1 is almost entirely intracellular in lymph node high endothelial venules, lymphatic sinus endothelium, and cultured endothelial cells but that a finite population, detectable only by fluorescent antibody or fluorescein-labeled (Fl)-acetylated low density lipoprotein uptake, cycles rapidly between the plasma membrane and EEA-1+ve (early endosome antigen 1) early endosomes. In addition, we show using full-length stabilin-1 cDNA and a stabilin-1/CD44 chimera in HeLa cells that intracellular targeting is influenced by the transmembrane domain/cytoplasmic tail, which contains a putative dileucine (DXXLL) Golgi to endosomal sorting signal. Finally, we provide evidence that the stabilin-1 Link domain binds neither hyaluronan nor other glycosaminoglycans. These properties support a role for stabilin-1 as a rapidly recycling scavenger receptor and argue against a role in cell adhesion or lymphocyte homing.

Selective alpha4beta7 integrin antagonists and their potential as antiinflammatory agents.

The accumulation of leukocytes in various tissues contributes to the pathogenesis of numerous human autoimmune diseases. The integrin alpha4beta7, expressed on the surface of B and T lymphocytes, plays an essential role in lymphocyte trafficking throughout the gastrointestinal (GI) tract via interaction with its primary ligand, mucosal addressin cell adhesion molecule (MAdCAM). Elevated MAdCAM expression in the intestines and liver has been linked to GI-associated autoimmune disorders, including Crohn's disease, ulcerative colitis, and hepatitis C. Monoclonal antibodies that block the interaction of alpha4beta7 with MAdCAM inhibit lymphocyte homing to murine intestines without effecting migration to peripheral organs; this suggests that alpha4beta7-selective antagonists might be useful as GI specific antiinflammatory agents. Here, we report the discovery of highly potent and selective alpha4beta7 antagonists affinity selected from a random peptide-phage library. Subsequent optimization of initial peptide leads afforded alpha4beta7-selective heptapeptide inhibitors that competitively inhibit binding to MAdCAM in vitro and inhibit lymphocyte homing to murine intestines in vivo. Substitution of a single carboxylate moiety alters selectivity for alpha4beta7 by more than 500-fold to afford a potent and selective alpha4beta1 antagonist. The antagonists described here are the first peptides to demonstrate potency and selectivity for alpha4beta7 compared to other integrins.

A 3D structure model of integrin alpha 4 beta 1 complex: I. Construction of a homology model of beta 1 and ligand binding analysis.

It is well established that integrin alpha 4 beta 1 binds to the vascular cell adhesion molecule (VCAM) and fibronectin and plays an important role in signal transduction. Blocking the binding of VCAM to alpha 4 beta 1 is thought to be a way of controlling a number of disease processes. To better understand how various inhibitors might block the interaction of VCAM and fibronectin with alpha 4 beta 1, we began constructing a structure model for the integrin alpha 4 beta 1 complex. As the first step, we have built a homology model of the beta 1 subunit based on the I domain of the integrin CD11B subunit. The model, including a bound Mg(2+) ion, was optimized through a specially designed relaxation scheme involving restrained minimization and dynamics steps. The native ligand VCAM and two highly active small molecules (TBC772 and TBC3486) shown to inhibit binding of CS-1 and VCAM to alpha 4 beta 1 were docked into the active site of the refined model. Results from the binding analysis fit well with a pharmacophore model that was independently derived from active analog studies. A critical examination of residues in the binding site and analysis of docked ligands that are both potent and selective led to the proposal of a mechanism for beta 1/beta 7 ligand binding selectivity.

Diamine containing VLA-4 antagonists.

Design and synthesis of a library as potential VLA-4 antagonists has been accomplished, based around a proposed pharmacophoric model. Compounds possessing submicromolar potency were identified and structure-activity relationships were seen across the library. Further derivatisation produced compounds with IC(50)'s <10 nmol for inhibiting the VLA-4 mediated binding of fibronectin to RAMOS cells, providing an ideal starting point for a lead optimisation Programme.

Evidence that ligand and metal ion binding to integrin alpha 4beta 1 are regulated through a coupled equilibrium.

We have used the highly selective alpha(4)beta(1) inhibitor 2S-[(1-benzenesulfonyl-pyrrolidine-2S-carbonyl)-amino]-4-[4-methyl-2S-(methyl-[2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl]-amino)-pentanoylamino]-butyric acid (BIO7662) as a model ligand to study alpha(4)beta(1) integrin-ligand interactions on Jurkat cells. Binding of [(35)S]BIO7662 to Jurkat cells was dependent on the presence of divalent cations and could be blocked by treatment with an excess of unlabeled inhibitor or with EDTA. K(D) values for the binding of BIO7662 to Mn(2+)-activated alpha(4)beta(1) and to the nonactivated state of the integrin that exists in 1 mm Mg(2+), 1 mm Ca(2+) were <10 pm, indicating that it has a high affinity for both activated and nonactivated integrin. No binding was observed on alpha(4)beta(1) negative cells. Through an analysis of the metal ion dependences of ligand binding, several unexpected findings about alpha(4)beta(1) function were made. First, we observed that Ca(2+) binding to alpha(4)beta(1) was stimulated by the addition of BIO7662. From solution binding studies on purified alpha(4)beta(1), two types of Ca(2+)-binding sites were identified, one dependent upon and the other independent of BIO7662 binding. Second, we observed that the metal ion dependence of ligand binding was affected by the affinity of the ligand for alpha(4)beta(1). ED(50) values for the metal ion dependence of the binding of BIO7762 and the binding of a lower affinity ligand, BIO1211, differed by 2-fold for Mn(2+), 30-fold for Mg(2+), and >1000-fold for Ca(2+). Low Ca(2+) (ED(50) = 5-10 microm) stimulated the binding of BIO7662 to alpha(4)beta(1). The effects of microm Ca(2+) closely resembled the effects of Mn(2+) on alpha(4)beta(1) function. Third, we observed that the rate of BIO7662 binding was dependent on the metal ion concentration and that the ED(50) for the metal ion dependence of BIO7662 binding was affected by the concentration of the BIO7662. These studies point to an even more complex interplay between metal ion and ligand binding than previously appreciated and provide evidence for a three-component coupled equilibrium model for metal ion-dependent binding of ligands to alpha(4)beta(1).

Novel sulfated lymphocyte homing receptors and their control by a Core1 extension beta 1,3-N-acetylglucosaminyltransferase.

L-selectin mediates lymphocyte homing by facilitating lymphocyte adhesion to addressins expressed in the high endothelial venules (HEV) of secondary lymphoid organs. Peripheral node addressin recognized by the MECA-79 antibody is apparently part of the L-selectin ligand, but its chemical nature has been undefined. We now identify a sulfated extended core1 mucin-type O-glycan, Gal beta 1-->4(sulfo-->6)GlcNAc beta 1-->3Gal beta 1-->3GalNAc, as the MECA-79 epitope. Molecular cloning of a HEV-expressed core1-beta 1,3-N-acetylglucosaminyltransferase (Core1-beta 3GlcNAcT) enabled the construction of the 6-sulfo sialyl Lewis x on extended core1 O-glycans, recapitulating the potent L-selectin-mediated, shear-dependent adhesion observed with novel L-selectin ligands derived from core2 beta1,6-N-acetylglucosaminyltransferase-I null mice. These results identify Core1-beta 3GlcNAcT and its cognate extended core1 O-glycans as essential participants in the expression of the MECA-79-positive, HEV-specific L-selectin ligands required for lymphocyte homing.

Identification of dual alpha 4beta1 integrin binding sites within a 38 amino acid domain in the N-terminal thrombin fragment of human osteopontin.

Previous work from our laboratory demonstrates that the alpha(4)beta(1) integrin is an adhesion receptor for OPN and that alpha(4)beta(1) binding site(s) are present in the N-terminal thrombin fragment of osteopontin (OPN) (Bayless, K. J., Meininger, G. A., Scholtz, J. M., and Davis, G. E. (1998) J. Cell Sci. 111, 1165-1174). The work presented here identifies two alpha(4)beta(1) binding sites within a recombinantly produced N-terminal thrombin fragment of human OPN. Initial experiments, using wild-type OPN containing an RGD sequence or an OPN-RGE mutant, showed identical alpha(4)beta(1)-dependent cell adhesive activity. A strategy to localize alpha(4)beta(1) binding sites within the thrombin fragment of osteopontin involved performing a series of truncation analyses. Removal of the last 39 amino acids (130) completely eliminated adhesion, indicating all binding activity was present within that portion of the molecule. Combined mutation and deletion analyses of this region revealed the involvement of dual alpha(4)beta(1) binding sites. Synthetic peptides for both regions in OPN, ELVTDFPTDLPAT (131) and SVVYGLR (162), were found to block alpha(4)beta(1)-dependent adhesion. The first peptide when coupled to Sepharose bound the alpha(4)beta(1) integrin directly whereas a mutated ELVTEFPTELPAT peptide showed a dramatically reduced ability to bind. These data collectively demonstrate that dual alpha(4)beta(1) integrin binding sites are present in a 38 amino acid domain within the N-terminal thrombin fragment of OPN.

Production of recombinant soluble human integrin alpha4beta1.

Integrin alpha4beta1 is a major leukocyte adhesion receptor that is a key target for the development of anti-inflammatory therapeutics. With the dual long-term goals of developing a reagent for use in high-throughput inhibitor screening assays and for crystallisation trials and subsequent structure determination, we have generated a recombinant soluble alpha4beta1 receptor. Both subunits were truncated prior to the transmembrane domains by site-directed mutagenesis and expressed using baculovirus infection of insect cells. The molecular weights of the recombinant subunits were as expected for post-translationally unmodified protein. In addition, as observed for the native subunit, a proportion of the alpha4 subunit was proteolytically processed into two fragments. ELISA and solid phase ligand-binding assays were performed to investigate the folding and functionality of the soluble integrin. The data suggest that the receptor was correctly folded and that it bound recombinant ligands with similar kinetics to the native molecule.

Combinatorial approaches to inhibitors of VLA-4: piperazine-peptoid-bisarylureas.

A combinatorial approach towards identifying inhibitors of VALA-4 was investigated. A library of piperazine-peptoid-bisarylureas was assembled in solid phase and screening in the novel v-well assay enabled the identification of active compounds.

Use of a novel fibronectin receptor for liver infiltration by a mouse lymphoma cell line RL-male1.

The mechanism whereby some lymphomas invade liver extensively has not been fully investigated. There is no basement membrane under the sinusoidal endothelium of the liver, and hepatocytes produce fibronectin (FN); therefore, adhesion to this FN may be particularly important for liver infiltration by lymphoma cells. A mouse lymphoma cell line, RL-male1, adhered to FN. However, this cell line did not express classical FN receptors such as very late antigen (VLA)-4 and VLA-5, as estimated by immunofluorescent staining. We have generated monoclonal antibodies (mAbs) that inhibit adhesion of RL-male1 cells to FN. Western blot and immunoprecipitation analyses showed that the new mAbs recognize a protein with an approximate molecular weight of 55,000 (p55). This antigenic protein was highly purified by immunoprecipitation and processed for microsequencing. From NH2-terminal sequence results, the p55 antigen was not identical to known FN receptors. Radioisotope-labeled RL-male1 cells, when injected i.v. into mice, rapidly infiltrated the liver (30-35% of injected cells), as measured by a gamma counter. Intravenous injection of the new mAbs partially (20%) blocked the infiltration of i.v.-injected lymphoma cells into the liver, whereas control rat IgG and an anti-CD11a mAb did not. These results demonstrate that the mouse lymphoma cell line RL-male1 nses a novel FN receptor for liver infiltration.

Crystal structure of a heparin- and integrin-binding segment of human fibronectin.

The crystal structure of human fibronectin (FN) type III repeats 12-14 reveals the primary heparin-binding site, a clump of positively charged residues in FN13, and a putative minor site approximately 60 A away in FN14. The IDAPS motif implicated in integrin alpha4beta1 binding is at the FN13-14 junction, rendering the critical Asp184 inaccessible to integrin. Asp184 clamps the BC loop of FN14, whose sequence (PRARI) is reminiscent of the synergy sequence (PHSRN) of FN9. Mutagenesis studies prompted by this observation reveal that both arginines of the PRARI sequence are important for alpha4beta1 binding to FN12-14. The PRARI motif may represent a new class of integrin-binding sites. The spatial organization of the binding sites suggests that heparin and integrin may bind in concert.

Mutational analysis of MAdCAM-1/alpha4beta7 interactions reveals significant binding determinants in both the first and second immunuglobulin domains.

The selective emigration of blood born leukocytes into tissues is mediated, in part by interactions of Ig-like cell adhesion molecules (IgCAMs) expressed on vascular endothelium and their cognate ligands, the leukocyte integrins. Within mucosal lymphoid tissues and gastrointestinal sites the mucosal vascular addressin. MAdCAM-1 is the predominant IgCAM, mediating specific lymphocyte homing via interactions with its ligand on lymphocytes, the integrin alpha4beta7. Previous studies have shown that an essential binding motif resides in the first Ig domain of all IgCAMs, containing an acidic residue (D or E) preceded by an aliphatic residue (L or I) that resides in strand C or the CD loop. However, domain swap experiments with MAdCAM-1 and VCAM-1 have shown a requirement for both Ig domains 1 and 2 for efficient integrin binding. We describe the use of chimeric MAdCAM-1/VCAM-1 receptors and point mutations in MAdCAM-1 to define other sites that are required for binding to the integrin alpha4beta7. We find that, in addition to critical CD loop residues, other regions in both domain one and two contribute to MAdCAM-1/alpha4beta7 interactions, including a buried arginine residue in the F strand of domain one and several acidic residues in a highly extended DE ribbon in domain 2. These mutations, when placed in the recently solved crystal structure of human MAdCAM-1 give insight into the integrin binding preference of this unique receptor.

Effects of tumor necrosis factor and lymphotoxin on peripheral lymphoid tissue development.

Previously, we have reported that neutralization of surface lymphotoxin (LT-alphabeta) in mice which expressed an LT-beta receptor-Fc fusion protein, driven by the cytomegalovirus promoter, resulted in an array of anatomic abnormalities. We now report that mice which express a tumor necrosis factor (TNF) receptor p60-Fc fusion protein (which neutralizes TNF and soluble LT-alpha3 activity) develop unique lymphoid abnormalities. Our data demonstrate that some aspects of peripheral lymphoid organ development require both surface LT-alphabeta and TNF interacting with their specific receptors. However, these related cytokines are also capable of signaling distinct developmental events. Splenic MAdCAM-1 expression, follicular dendritic cell localization and normal Peyer's patch development all require both surface LT-alphabeta and TNF activity. Marginal zone formation and splenic B cell localization primarily require surface LT-alphabeta-LT-beta receptor interactions. Primary follicle formation was dependent upon TNF receptor(s) engagement. Interestingly spleen, lymph nodes and Peyer's patches from TNF receptor p60-Fc-expressing mice all develop different abnormalities, suggesting distinct pathways of development in these lymphoid organs. Thymus development appears to be independent of these signaling pathways. These results demonstrate that TNF and LT are crucial for normal peripheral, but not central lymphoid organ development.

Role of two conserved glycine residues in the beta-propeller domain of the integrin alpha4 subunit in VLA-4 conformation and function.

The N-terminal region of the alpha integrin subunits is predicted to fold into a beta-propeller domain. Using K562 alpha4 transfectants we show that mutations at alpha4 subunit residues Gly130 and Gly190 affect the conformation of this domain causing a reduction in the recognition of alpha4 by anti-alpha4 antibodies which map to the beta-propeller. The improper alpha4 conformation also led to an altered association with the beta1 subunit, and to a lack of alpha4beta1 adhesion to VCAM-1 and CS-1/fibronectin, as well as an abolishment of anti-alpha4- and anti-beta1-dependent homotypic aggregation. The total conservation of Gly130 and Gly190 among integrin alpha subunits suggests their importance in the correct folding of their respective beta-propeller domains, and thus, in the adhesive activity of the integrins.

The structure of immunoglobulin superfamily domains 1 and 2 of MAdCAM-1 reveals novel features important for integrin recognition.

BACKGROUND: Mucosal addressin cell adhesion molecule 1 (MAdCAM-1) is a cell adhesion molecule that is expressed on the endothelium in mucosa, and guides the specific homing of lymphocytes into mucosal tissues. MAdCAM-1 belongs to a subclass of the immunoglobulin superfamily (IgSF), the members of which are ligands for integrins. Human MAdCAM-1 has a unique dual function compared to other members in the same subclass in that it binds both the integrin alpha4beta7, through its two IgSF domains, and a selectin expressed on leukocytes, via carbohydrate sidechains. The structure determination of the two IgSF domains and comparison to the N-terminal two-domain structures of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecules (ICAM-1 and ICAM-2) allow us to assess the molecular basis of the interactions between integrins and their preferred ligands. RESULTS: The crystal structure of a fragment containing the two IgSF domains of human MAdCAM-1 has been determined to 2.2 A resolution. The structure of MAdCAM-1 reveals two separate integrin-recognition motifs. The key integrin-binding residue, Asp42, resides in the CD loop of domain 1; a buried arginine residue (Arg70) plays a critical role in maintaining the conformation of this loop. The second binding site is associated with an unusual long D strand in domain 2. The D and E strands extend beyond the main body of the domain, forming a negatively charged beta ribbon unique to MAdCAM-1. This ribbon is located on the same face as the key aspartate residue in domain 1, consistent with evidence that it is involved in integrin binding. CONCLUSIONS: The structural comparison of MAdCAM-1 to other members of the same IgSF subclass reveals some interesting features. Firstly, MAdCAM-1, like VCAM-1, has the key integrin-binding residue located on the protruding CD loop of domain 1 and binds to an integrin that lacks an I domain. This is in contrast to ICAM-1 and ICAM-2 where the key residue is located at the end of the C strand on a flat surface and which bind to integrins that contain I domains. Secondly, architectural differences in the CD loops of MAdCAM-1 and VCAM-1 cause an 8 A shift in position of the critical aspartate residue, and may partly determine their binding preference for different integrins. Finally, the unusual charge distribution of the two-domain fragment of MAdCAM-1 is predicted to orient the molecule optimally for integrin binding on the top of its long mucin-like stalk.

Identification of ligand binding sites on integrin alpha4beta1 through chemical cross-linking.

We have used chemical cross-linking to identify sequences in integrin alpha4beta1 that are involved in its interactions with ligands. A recently described leucine-aspartic acid-valine (LDV)-based small molecule inhibitor of alpha4beta1 (BIO-1494), that contained a single reactive amino group for targeting the cross-linking, was used for these studies. The specificity of the interaction was defined by (i) the ability to block the interaction with a competitive inhibitor lacking the reactive group, (ii) the absolute requirement of divalent cations for cross-linking, and (iii) the lack of cross-linking to the functionally related integrin alpha4beta7. With ANB-NOS as the cross-linker, only the beta1 chain was labeled with BIO-1494, while with the more flexible cross-linker DSS both the alpha4 and beta1 chains were modified. Similar results were obtained when cross-linking was performed on K562 cells expressing alpha4beta1 but not on K562 cells expressing alpha2beta1. The site of cross-linking on the beta1 chain was localized by CNBr peptide mapping within residues 130-146, a region that contains the putative metal binding site DXSXS and for which analogous data had been generated with RGD binding to integrin alphaIIbbeta3. The striking similarity between the data we generated for an LDV ligand and published data for the RGD family supports the notion of a common ligand binding pocket formed by both integrin chains. The cross-linking strategy developed here should serve as a useful tool for studying alpha4beta1 function.

A role of chondroitin sulfate glycosaminoglycan binding site in alpha4beta1 integrin-mediated melanoma cell adhesion.

We have previously reported that alpha4beta1 (but not alpha5beta1) integrin-mediated melanoma cell adhesion is inhibited by removal of cell surface chondroitin sulfate glycosaminoglycan (CSGAG), suggesting that melanoma chondroitin sulfate proteoglycan plays a role in modulating the adhesive function of alpha4beta1 integrin. In the current study, we demonstrated that alpha4beta1 integrin binds to CSGAG. We have identified a peptide from within alpha4 integrin termed SG1 (KKEKDIMKKTI) that binds to cell surface melanoma chondroitin sulfate proteoglycan, indicating that SG1 represents a CSGAG binding site within the alpha4 integrin subunit. Soluble SG1 inhibits alpha4beta1 integrin-mediated human melanoma cell adhesion to CS1. Polyclonal antibody generated against the peptide inhibits melanoma cell adhesion to CS1, and the inhibition is reversed by Mn2+ and an activating monoclonal antibody anti-beta1 (8A2). Additionally, pretreatment of cells with anti-SG1 IgG inhibits the expression of the monoclonal antibody 15/7 epitope in the presence of soluble CS1 peptide, suggesting that anti-SG1 IgG prevents ligand binding by alpha4beta1 integrin. These results demonstrate that alpha4beta1 integrin interacts directly with CSGAG through SG1 site, and that this site can affect the ligand binding properties of the integrin.