Binding between the Integrin αXβ2 (CD11c/CD18) and Heparin

The interactions between cell surface receptors and sulfated glucosamineglycans serve ubiquitous roles in cell adhesion and receptor signaling. Heparin, a highly sulfated polymer of uronic acids and glucosamine, binds strongly to the integrin receptor αXβ2 (p150,95, CD11c/CD18). Here, we analyze the structural motifs within heparin that constitute high affinity binding sites for the I domain of integrin αXβ2. Heparin oligomers with chain lengths of 10 saccharide residues or higher provide strong inhibition of the binding by the αX I domain to the complement fragment iC3b. By contrast, smaller oligomers or the synthetic heparinoid fondaparinux were not able to block the binding. Semipurified heparin oligomers with 12 saccharide residues identified the fully sulfated species as the most potent antagonist of iC3b, with a 1.3 μm affinity for the αX I domain. In studies of direct binding by the αX I domain to immobilized heparin, we found that the interaction is conformationally regulated and requires Mg2+. Furthermore, the fully sulfated heparin fragment induced conformational change in the ectodomain of the αXβ2 receptor, also demonstrating allosteric linkage between heparin binding and integrin conformation. The interactions between cell surface receptors and sulfated glucosamineglycans serve ubiquitous roles in cell adhesion and receptor signaling. Heparin, a highly sulfated polymer of uronic acids and glucosamine, binds strongly to the integrin receptor αXβ2 (p150,95, CD11c/CD18). Here, we analyze the structural motifs within heparin that constitute high affinity binding sites for the I domain of integrin αXβ2. Heparin oligomers with chain lengths of 10 saccharide residues or higher provide strong inhibition of the binding by the αX I domain to the complement fragment iC3b. By contrast, smaller oligomers or the synthetic heparinoid fondaparinux were not able to block the binding. Semipurified heparin oligomers with 12 saccharide residues identified the fully sulfated species as the most potent antagonist of iC3b, with a 1.3 μm affinity for the αX I domain. In studies of direct binding by the αX I domain to immobilized heparin, we found that the interaction is conformationally regulated and requires Mg2+. Furthermore, the fully sulfated heparin fragment induced conformational change in the ectodomain of the αXβ2 receptor, also demonstrating allosteric linkage between heparin binding and integrin conformation. Increasing evidence points to an important function of heparin in the immune system. Heparin is exclusively synthesized by connective tissue mast cells and released from storage granula in the inflammatory responses mediated by these leukocytes. Furthermore, several receptors on leukocytes are able to bind with high affinity to heparin. These include the β2 integrins αMβ2 (Mac-1, CD11b/CD18) and αXβ2 (p150,95, CD11c/CD18) (1Coombe D.R. Watt S.M. Parish C.R. Blood. 1994; 84: 739-752Crossref PubMed Google Scholar, 2Diamond M.S. Alon R. Parkos C.A. Quinn M.T. Springer T.A. J. Cell Biol. 1995; 130: 1473-1482Crossref PubMed Scopus (253) Google Scholar), which play key roles in the adhesion, migration, and binding of complement fragments by myeloid leukocytes. αMβ2 and αXβ2 integrins, also referred to as complement receptors 3 and 4, respectively, bind strongly to a protelytic fragment of complement factor 3 designated iC3b, as shown by both cellular and biochemical assays. iC3b plays an important role in phagocytic uptake of microbes by leukocytes of the myeloid lineages. Diamond et al. (2Diamond M.S. Alon R. Parkos C.A. Quinn M.T. Springer T.A. J. Cell Biol. 1995; 130: 1473-1482Crossref PubMed Scopus (253) Google Scholar) reported the adhesion of neutrophil granulocytes to heparin-coated surfaces through the αMβ2 integrin, which is abundantly expressed on these leukocytes. The αXβ2 integrin, primarily expressed on monocytes, macrophages, and dendritic cells, was also demonstrated to support adhesion to heparin by use of cell line transfectants (2Diamond M.S. Alon R. Parkos C.A. Quinn M.T. Springer T.A. J. Cell Biol. 1995; 130: 1473-1482Crossref PubMed Scopus (253) Google Scholar). Integrin receptors contain multiple domains in their ectodomain. β2 integrins, which in addition to αMβ2 and αXβ2 include the αLβ2 integrin (LFA-1, CD11a/CD18), bind ligands through an inserted (I) domain in the α subunit. Previous studies have indicated a central role for the αM and αX I domains in binding to heparin (2Diamond M.S. Alon R. Parkos C.A. Quinn M.T. Springer T.A. J. Cell Biol. 1995; 130: 1473-1482Crossref PubMed Scopus (253) Google Scholar) and shown that the affinity of the αXI domain for heparin is significantly higher than the affinity of the αM I domain (3Vorup-Jensen T. Carman C.V. Shimaoka M. Schuck P. Svitel J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1614-1619Crossref PubMed Scopus (80) Google Scholar). In the metal ion-dependent adhesion site (MIDAS) 2The abbreviations used are: MIDAS, metal ion-dependent adhesion site; RU, arbitrary response unit(s); MES, 4-morpholineethanesulfonic acid; TBS, Tris-buffered saline.2The abbreviations used are: MIDAS, metal ion-dependent adhesion site; RU, arbitrary response unit(s); MES, 4-morpholineethanesulfonic acid; TBS, Tris-buffered saline. of the I domain, a Mg2+ ion forms a crucial bond to an acidic residue in protein ligands. However, the requirement for Mg2+ in the binding between integrin I domains and heparin is unclear. For many protein ligands the binding to integrin I domains is regulated through conformational changes, where the open conformation of the I domain binds these ligands with several magnitudes stronger affinity than the closed conformation (4Shimaoka M. Takagi J. Springer T.A. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 485-516Crossref PubMed Scopus (435) Google Scholar). By contrast, conformational regulation of the binding by I domains to heparin or other nonproteinous ligands has not been studied. The structure of heparin has been subject to considerable investigation. Heparin is a sulfated, linear polysaccharide with a repeating disaccharide residue of d-glucosamine and uronic acids. Each repeating residue of glucosamine and uronic acid may hold a maximum of three sulfo groups, but other, less sulfated disaccharides can also be isolated from enzymatically degraded heparin. This microheterogeneity and the polydisperse length with the average Mr of natural heparin chains distributed between 10,000 and 12,000 constitute together a highly complex structure (5Hileman R.E. Fromm J.R. Weiler J.M. Linhardt R.J. Bioessays. 1998; 20: 156-167Crossref PubMed Scopus (507) Google Scholar, 6Capila I. Linhardt R.J. Angew. Chem. Int. Ed. Engl. 2002; 41: 391-412Crossref PubMed Scopus (1504) Google Scholar, 7Linhardt R.J. J. Med. Chem. 2003; 46: 2551-2564Crossref PubMed Scopus (424) Google Scholar). No studies on integrins have addressed the character of the binding sites in heparin for I domains. These properties are important in understanding the ability to bind pharmacologically important glucosamineglycans such as fondaparinux sodium, a synthetic heparinoid recently marketed for antithrombotic treatment (8de Kort M. Buijsman R.C. van Boeckel C.A. Drug Discov. Today. 2005; 10: 769-779Crossref PubMed Scopus (78) Google Scholar). Here we analyze the binding between heparin and the αXI domain and identify oligomers with 10 saccharide residues or more as potent ligands for the αX I domain. The binding to heparin was dependent on Mg2+ and conformationally regulated similar to what has been reported for protein ligands. Fully sulfated oligomers bound the αX I domain with the highest affinity and with sufficient potency to induce conformational change in the ectodomain of αXβ2 integrin. Preparation and Characterization of Bovine Lung Heparin Oligosaccharides—The heparin oligosaccharide mixture, prepared from bovine lung heparin (Sigma) by controlled enzymatic depolymerization with heparin lyase I (EC 4.2.2.7; IBEX, Montreal, Canada), was fractionated by gel permeation chromatography on a P-10 column (Bio-Rad) to obtain oligosaccharides uniform in size. The fraction consisting of dodecasaccharides was further separated strong high chromatography on a column with a linear from to a of A. Linhardt R.J. 1995; PubMed Scopus Google Scholar). and were and The and of the oligosaccharide were by on with a linear from 12 to by and with a of heparin oligosaccharide R.E. A. Weiler J.M. J. Linhardt R.J. J. Sci. PubMed Scopus Google Scholar). I and for the with and of αM and αX I domains was (3Vorup-Jensen T. Carman C.V. Shimaoka M. Schuck P. Svitel J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1614-1619Crossref PubMed Scopus (80) Google Scholar, R. M. T. J. Biol. Chem. PubMed Scopus Google Scholar, T. Shimaoka M. U. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). In the αX I domain and open conformation αM and αXI domains the and respectively, were expressed in and from the The affinity of the open conformation αM and αX I domains for heparin and heparin fragments was by inhibition of I domain binding to The were by with a in cells with arbitrary response of of cell of iC3b and with a cell with in as T. Shimaoka M. U. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). The for the binding between the open conformation αX I domain and the immobilized iC3b was as T. Shimaoka M. U. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar) from the to the response In is the of I domain, and is the response For inhibition with the αX I domain, bovine heparin fondaparinux and heparin were from to with a of or μm of the αX I domain in with 10 10 MES, the αM I domain a of μm was with the in with 10 The of I domain and heparin were the and surfaces with a of by a of and in MES, The of the interaction between I domain and heparin in was as the binding in to iC3b by an of the affinity of the interaction between heparin oligomers and the αX I domain, we the of the heparin oligomers on the of binding between the I domain and immobilized iC3b. The for the and of the I domain binding to iC3b are to the response the through In is the the where the to the response from of from surface can be and with α as the of the and can be to the The of the a as which the on of the linear between and the of to the of I domain with was by as a function of of heparin The direct binding of I domains to heparin was by heparin to with a surface with The surface was with a and with and of of by of sites by of heparin with an average of and a was in 10 and the surface to for through the of the heparin. The bond from the between and the is not in and the bond was to a bond by of of by of the surface with of of 10 This immobilized heparin a to The binding to the surfaces was for the αM and αX I domains with the that the was of in the β2 of ability of heparin and fragments of heparin to induce conformational change in the αXβ2 integrin was by use of the A. S. J. M. J. PubMed Scopus Google Scholar), which an in the of the chain M. Takagi J. Springer T.A. J. PubMed Scopus Google Scholar), in the of an with αXβ2. αLβ2 and αXβ2 integrins with a were expressed in cell and as Shimaoka M. T. Springer T.A. PubMed Scopus Google Scholar). The were in and were with the as by Takagi et al. J. Springer T.A. Struct. Biol. PubMed Scopus Google Scholar). The were with of in by in with and with bovine (Sigma) in in of to a of in with were to and for by three in The with immobilized integrins were with heparin, bovine heparin, or fondaparinux in between and in with and For immobilized αXβ2 and αLβ2 integrins were also with heparin in with and or in with and or in with or in with and μm U. J. Biol. Chem. 2002; PubMed Scopus Google Scholar). The were for to or to in with bovine were in the for The were with and the addition of The of the αX I with Heparin enzymatic depolymerization with heparin lyase is to from heparin a of oligomers with oligomers in lengths from a of saccharide residues to saccharide residues with a Mr between and respectively, as of the open conformation αXI domain binding to iC3b. The αX I domain binds the iC3b fragment of complement factor 3 with a of μm with the αX I domain to to heparin oligosaccharides and binding to iC3b, the and oligomers a on the response of was used to binding to iC3b which in the of inhibition bound and of αX I domain μm was The to obtain a in the response was and μm for the and and heparin is a potent of binding by the open conformation αX I domain to immobilized iC3b an average Mr of and of the of heparin to obtain inhibition was inhibition of the binding by I domains to immobilized iC3b with heparin and heparin In the of the average Mr of heparin was as the Mr of smaller fragments was from a Mr of inhibition of the binding by the open conformation αX I domain to immobilized iC3b as a function of the of heparin with a of I domain μm were with heparin, and the response was the of the The of inhibition was to the response in the of heparin. The binding by the αX to iC3b in the of in from to inhibition of the binding by the αX I domain to immobilized iC3b with the from ion chromatography of the heparin and were from to For the of fondaparinux on the binding to iC3b is The as in is expressed as a of from the similar to the in inhibition of the binding by the αM to iC3b in the of is shown for from to on the of the binding by αX I domain to iC3b by heparin and heparin the as the of the to the for the binding of the αM and αX I domains to iC3b shown in and as function of the I domain was from a line with to the the in as function of the heparin for and bovine heparin in a the in as a function of the of oligomers in a from to the for the inhibition of I domain binding to iC3b with heparin in The were from the of oligosaccharides the response the of the by or by a in The for the open conformation αM and αX I domain for heparin were in an (3Vorup-Jensen T. Carman C.V. Shimaoka M. Schuck P. Svitel J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1614-1619Crossref PubMed Scopus (80) Google Scholar) from with immobilized The αM I domain, to the αX I domain to the open conformation R. M. T. J. Biol. Chem. PubMed Scopus Google Scholar), bound iC3b with a of μm in with (3Vorup-Jensen T. Carman C.V. Shimaoka M. Schuck P. Svitel J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1614-1619Crossref PubMed Scopus (80) Google Scholar, R. M. T. J. Biol. Chem. PubMed Scopus Google Scholar). No inhibition was from of the heparin oligomers to the binding by the open conformation αM I domain to immobilized iC3b and The interaction between heparin oligomers and the αX and αM I domains was further by the of the oligomers on the as has been for other inhibition with use of D.R. S. M. J. J. Biol. Chem. 2005; PubMed Scopus Google Scholar). the of 3 and we the I domain for from to μm and the shown by the in was a linear between the I domain and with to that the binding was not by Furthermore, from the of the line of and of as from was to be which is within of of T. Shimaoka M. U. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). are significantly 10,000 R. J. PubMed Scopus Google Scholar) and the linear between and the I domain we that the of binding are not by the of the heparin oligomers and heparin on the The heparin to the for and were and The of the αX I for Heparin further the interaction between heparin and the αX I domain, we the oligomers by strong high The oligomers were separated in and species by and to dodecasaccharides on with oligosaccharide R.E. A. Weiler J.M. J. Linhardt R.J. J. Sci. PubMed Scopus Google Scholar). to by A. Linhardt R.J. 1995; PubMed Scopus Google Scholar), fraction of higher than to a fully sulfated, with a maximum of sulfo groups, with less than sulfo The and oligomers with fraction in addition to highly sulfated were as for the and and the were by the to the response the of the by The oligomers bound the αX I domain with the highest affinity with an of 1.3 μm The less sulfated oligomers bound with an of the and a affinity than the with the in fraction of The fraction the potency in the binding between the αX I domain and iC3b, the an affinity to the affinity of oligomers and These were further by the by oligomers of the in binding by the αX I domain and The the of the was to that for the inhibition of the response with the most potent for the αX I domain. For with the heparin we fondaparinux sodium, which is a synthetic with a Mr of and a of sulfo (8de Kort M. Buijsman R.C. van Boeckel C.A. Drug Discov. Today. 2005; 10: 769-779Crossref PubMed Scopus (78) Google Scholar), less than the maximum of sulfo similar with used for heparin fragments or the fondaparinux was not able to the binding of the αX I domain to iC3b of the αX and αM I to direct binding of open and closed αX and αM I domains to heparin was by acid depolymerization of heparin oligomers with a that for to surfaces J. PubMed Scopus Google Scholar). with heparin the average Mr was from 10,000 to with a of by The open conformation αX I domain a of μm bound to heparin in the of Mg2+ binding by the open conformation αX I domain. The conformational regulation of the binding was by the binding of the αX I domain a of In a response was a affinity for heparin with the that heparin in is a of the binding by the open conformation αM I domain to iC3b, direct binding of domain to immobilized heparin was the domain was a of μm in the of Mg2+ of in the αXβ2 by by the β2 A. S. J. M. J. PubMed Scopus Google Scholar) is a for the structural within the that the β2 integrins from a conformation to the conformation M. Takagi J. Springer T.A. J. PubMed Scopus Google Scholar). αXβ2 integrin was immobilized in and with the The binding of the a with to an of μm Heparin, and fondaparinux a in but were less potent than the not change the a of also the with other to the conformation of β2 In with Mg2+ and the αXβ2 integrin in a with a of the The addition of a of conformational change and of binding in integrins, is a antagonist of β2 integrin binding that through allosteric regulation of the β2 chain U. J. Biol. Chem. 2002; PubMed Scopus Google Scholar, Shimaoka M. A. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). reported for αLβ2 integrin Shimaoka M. A. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) induced in the αXβ2 integrin with the that the conformation of αXβ2 integrin Shimaoka M. T. Springer T.A. PubMed Scopus Google Scholar). The addition of a binding a with that for the of or to the αLβ2 integrin in the with the or with heparin, heparin or not of the to the β2 chain that the of αLβ2 and αXβ2 integrin immobilized in the were not In we analyze the binding between heparin and αXβ2 integrin and that the αX I domain has high affinity for fully sulfated heparin oligomers with a length of 12 a conformational regulation of the binding between heparin and the αXβ2 integrin, which the role of conformational regulation in integrin binding to a Furthermore, the of the binding between heparin oligomers and the receptor is sufficient to the αXβ2 integrin as shown by The αXβ2 integrin with several domains The I domain of the α chain is the binding for several protein ligands such as and iC3b. has identified the I domain as also a binding domain for heparin (3Vorup-Jensen T. Carman C.V. Shimaoka M. Schuck P. Svitel J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1614-1619Crossref PubMed Scopus (80) Google Scholar). The I domain may referred to as the and reported the open conformation αX I domain binds strongly to heparin (3Vorup-Jensen T. Carman C.V. Shimaoka M. Schuck P. Svitel J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1614-1619Crossref PubMed Scopus (80) Google Scholar), but a of the binding in heparin has not been In the we provide a more of the interaction between heparin and the αX I domain and the of the interaction through the ability of the heparin oligomers to the binding of the open conformation αX I domain to iC3b as by The of inhibition was by the response the of the in the or or heparin However, was not for we also the of heparin oligomers on the in the binding between the αX I domain and we found between the that the from these are a of the of binding between the heparin oligomers and the I domain. and of heparin oligomers that the αX I domain bound and oligomers with an affinity to a of By contrast, oligomers bound the I domain with affinity to an of that a of 12 saccharide residues is to obtain high affinity binding between the αX I domain and heparin. with these a synthetic heparin that is used in treatment as for (8de Kort M. Buijsman R.C. van Boeckel C.A. Drug Discov. Today. 2005; 10: 769-779Crossref PubMed Scopus (78) Google Scholar), interaction with the αX I domain. of the oligomers oligomers with of of the ability of these oligomers to the binding of the open conformation αX I domain that the most sulfated of the the highest affinity for the αX I domain. studies have shown that binding by αMβ2 and αXβ2 integrins to heparin is dependent on (2Diamond M.S. Alon R. Parkos C.A. Quinn M.T. Springer T.A. J. Cell Biol. 1995; 130: 1473-1482Crossref PubMed Scopus (253) Google Scholar). However, both receptors contain multiple and sites in their which on which metal were for the binding to heparin. The integrin I domain metal site (4Shimaoka M. Takagi J. Springer T.A. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 485-516Crossref PubMed Scopus (435) Google Scholar), which has a stronger affinity for Mg2+ than for and of these primarily is with Mg2+ 1998; PubMed Scopus Google Scholar). T. and T. Biophys. a the for Mg2+ but not for In the the Mg2+ ion the chains of and The metal ion is in the of the site and a chain in protein ligands (4Shimaoka M. Takagi J. Springer T.A. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 485-516Crossref PubMed Scopus (435) Google Scholar). the site in the αX I domain was in heparin binding by heparin to a surface and in the open conformation I domain in the of Mg2+ or of the I domain to immobilized heparin Mg2+. have been in several to to the binding between heparin and protein binding to heparin has been less heparin J. Linhardt R.J. 10: PubMed Scopus Google Scholar). that the heparin binding by the αX I domain is a of a Mg2+ ion to The length of heparin from S. Biophys. J. 2003; PubMed Scopus Google Scholar) or direct on heparin in complex with by J. Biol. Chem. 2005; PubMed Scopus Google Scholar) to oligomers be to a length of which is with the of the I domain T. Shimaoka M. U. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). that interactions on the αX I domain surface and heparin to the binding. were reported for other Linhardt R.J. Struct. Biol. PubMed Scopus Google Scholar), where the binding of heparin to protein surfaces is by the induced by the of the heparin sulfo In is of that of the heparin oligomers identified the sulfated as the of the αX I domain and that binding of the I domain to heparin Mg2+. studies on the αX I domain identified a of or residues of a length of by residues and the with the Mg2+ ion T. Shimaoka M. U. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). of the αXI domain to protein ligands is as we for binding to heparin. This that the the binding for heparin, as shown by for other I domains bound to protein ligands (4Shimaoka M. Takagi J. Springer T.A. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 485-516Crossref PubMed Scopus (435) Google Scholar). the of residues in an through the a structural that for binding such as heparin, and that a length of saccharide is to obtain a affinity is in with the of the of the site to be fully with the we that heparin, on average to a has a higher affinity for the αX I domain than the for is to to the of multiple binding sites within an the of heparin. have the for heparin on the of the but the and binding the of binding sites is by However, as a for is we have reported the of the interactions in of the of The αM I domain not have the of residues the as is found on the αX I domain in of heparin bound with affinity to the open conformation αM I domain than to the αX I domain. The that is not by the length of the heparin. studies on integrin receptors have the of affinity regulation in the binding by these receptors through conformational in the receptor ectodomain C.V. Springer T.A. Cell Biol. 2003; PubMed Scopus Google Scholar). In the I domain, the conformational regulation is with the ability of the Mg2+ ion to acidic chains of protein ligands. the binding to immobilized heparin by the αXI domain, which we have shown the closed conformation with a affinity for protein ligands T. Shimaoka M. U. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). with the open conformation αX I domain, binding by the domain was evidence of the of integrin conformation was by the of an in the β2 chain of immobilized αXβ2 integrin. binding by integrins is with a change in the conformation of ectodomain of the in the the receptor a a change to the conformation the receptor for binding In the of β2 integrins, an in the chain by the is the receptor is found in the conformation Shimaoka M. T. Springer T.A. PubMed Scopus Google Scholar). By the αXβ2 integrin immobilized in with the we were able to strongly induce the of the with Integrin receptors between the and J. T. Springer T.A. 2002; PubMed Scopus Google Scholar), and of an of a strong the heparin be to the of integrin of the conformation. that the conformation of the αX I domain binding by heparin that binding of heparin the conformation of αXβ2. J. Svitel for for on surface

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