Production and properties of a bifunctional fusion protein that mediates attachment of vero cells to cellulosic matrices

The sequence Arg-Gly-Asp (RGD) in extracellular matrix proteins such as fibronectin, collagen, and laminin mediates cell attachment by interacting with proteins of the integrin family of cell surface receptors. A gene fusion encoding the RGD-containing peptide, fused to the C-terminus of a cellulose-binding domain (CBD/RGD), was expressed in Escherichia coli. Cultures produced up to 50 mg of CBD/RGD per liter, most of which was extracellular. It was purified from the culture supernatant by affinity chromatography on cellulose. CBD/RGD promoted the attachment of green monkey Vero cells to polystyrene and cellulose acetate. Attachment was inhibited by small synthetic peptides containing the RGD sequence. CBD/RGD was as effective as collagen in promoting the attachment of Vero cells to Cellsnowtrade mark microcarriers. (c) 1995 John Wiley & Sons, Inc.     

Structural and functional aspects of decorsin and its analog as recognized by integrin αIIbβ3

Decorsin is an antagonist of platelet glycoprotein integrin αIIbβ3 on platelets; the protein is 39 amino acids long with three disulfide bridges in its tertiary structure. To demonstrate decorsin’s mechanism of action, we applied the computational virtual technique and platelet aggregation inhibition assay, which showed that the flanking amino-acid residues of the Arg–Gly–Asp (RGD) motif play an important role in platelet aggregation. The computational simulations revealed that the RGD motif mainly contributes to the stability of the complex when decorsion interacts with integrin αIIbβ3. However, the C-terminal residues, such as 34A→W and 35D→R, was also found to possibly play a key role in their binding structures. Moreover, we produced a decorsin analog (A34W plus D35R decorsin), in which the 34A (alanine) and 35D (aspartic acid) residues were respectively substituted by W (tryptophan) and R (arginine). This isoform was then recombinantly expressed in Escherichia coli. Intriguingly, this mutant type showed higher anti-platelet aggregation activity than the wildtype. Our study may further contribute to finding decorsin mutants with higher anti-platelet aggregation activity.     

The RGD containing site of the mouse laminin A chain is active for cell attachment, spreading, migration and neurite outgrowth

The laminin A chain has been sequenced by cDNA cloning and was found to contain an RGD sequence. Synthetic peptides containing the RGD sequence and flanking amino acids were active in mediating cell adhesion, spreading, migration, and neurite outgrowth. Furthermore, endothelial cell attachment to a laminin substrate was inhibited by an RGD-containing synthetic peptide. Antisera against the integrin (fibronectin) receptor, and monoclonal antibody to the integrin, VLA-6, inhibited cell interaction with laminin, as well as with peptides containing an RGD sequence. These results suggest that the RGD containing site of laminin is active and interacts with the integrin family of receptors in certain cells.     

Asia1型口蹄疫病毒受体结合位点多样性

【目的】口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)通过结构蛋白VP1 G-H环上高度保守的精氨酸-甘氨酸-天门冬氨酸(Arg-Gly-Asp,RGD)基序与整联蛋白结合起始病毒的感染,但FMDV是RNA病毒,在环境条件变化时,FMDV能够以非RGD的途径起始病毒的感染。为了研究FMDV Asia1/JS/China/05田间舌皮毒经两种不同的途径短期传代后细胞受体结合基序RGD的变异。【方法】采用RT-PCR方法扩增FMDV Asia1/JS/China/05田间毒、田间毒的乳鼠适应毒第四代(MF4)和接种田间毒的牛同居感染的猪水泡病料适应细胞的第八代毒(PBF8)结构蛋白VP1基因,并对不同病毒VP1基因的PCR产物测序和cDNA文库测序。【结果】以含RGD受体结合基序为优势的田间毒在乳鼠上短期传代后出现了含精氨酸-丝氨酸-天门冬氨酸(Arg-Ser-Asp,RSD)和RGD受体结合基序的混合种群,而同居感染后的细胞传代病毒种群则以含精氨酸-天门冬氨酸-天门冬氨酸(Arg-Asp-Asp,RDD)受体结合基序为优势种群。【结论】发现了含RGD受体结合位点为优势的FMDV种群,经不同的宿主短期传代后产了含RSD或RDD受体结合基序的优势种群,该发现不仅增加了保守基序RGD发生替换的FMDV变异株的数量,而且为FMDV的细胞识别和宿主嗜性的改变等进一步研究奠定了物质基础。     

Interaction of integrins alpha v beta 3 and glycoprotein IIb-IIIa with fibrinogen. Differential peptide recognition accounts for distinct binding sites

Glycoprotein (GP) IIb-IIIa is the major fibrinogen receptor on platelets and participates in platelet aggregation at the site of a wound. Integrin alpha v beta 3, which contains an identical beta-subunit, is expressed on endothelial cells and also serves as a fibrinogen receptor. Here, we demonstrate by several criteria that purified GPIIb-IIIa and integrin alpha v beta 3 bind to distinct sites on fibrinogen. First, a plasmin-generated fragment of fibrinogen lacking the RGD sequence at residues 572-574 retained the ability to bind GPIIb-IIIa, but failed to bind integrin alpha v beta 3. Second, a monoclonal antibody which exclusively recognizes the RGD sequence at fibrinogen A alpha chain residues 572-574 abolished interaction between integrin alpha v beta 3 and fibrinogen, but had only a minimal effect on fibrinogen binding to GPIIb-IIIa. Finally, we show that the difference in recognition of sites on fibrinogen by these two integrins is probably a consequence of their remarkably different ligand binding properties. Peptides corresponding to fibrinogen gamma chain residues 400-411 effectively blocked RGD sequence and fibrinogen binding by GPIIb-IIIa, but had no effect on the ability of integrin alpha v beta 3 to bind these ligands. We also show that integrin alpha v beta 3 has a higher affinity than GPIIb-IIIa for a synthetic hexapeptide containing the RGD sequence. In fact, this RGD-containing peptide was 150-fold more effective at blocking fibrinogen binding to integrin alpha v beta 3 than to GPIIb-IIIa. Collectively, our results demonstrate that integrins alpha v beta 3 and GPIIb-IIIa display qualitative and quantitative differences in their ligand binding properties, as is evident by their ability to interact with synthetic peptides. The ultimate result of these differences is the recognition of distinct sites on fibrinogen by the two integrins. These observations may have relevance in the processes of hemostasis and wound healing.     

Specificity of the VP1 GH loop of Foot-and-Mouth Disease virus for alphav integrins

Foot-and-mouth disease virus (FMDV) can use a number of integrins as receptors to initiate infection. Attachment to the integrin is mediated by a highly conserved arginine-glycine-aspartic acid (RGD) tripeptide located on the GH loop of VP1. Other residues of this loop are also conserved and may contribute to integrin binding. In this study we have used a 17-mer peptide, whose sequence corresponds to the GH loop of VP1 of type O FMDV, as a competitor of integrin-mediated virus binding and infection. Alanine substitution through this peptide identified the leucines at the first and fourth positions following RGD (RGD+1 and RGD+4 sites) as key for inhibition of virus binding and infection mediated by alphavbeta6 or alphavbeta8 but not for inhibition of virus binding to alphavbeta3. We also show that FMDV peptides containing either methionine or arginine at the RGD+1 site, which reflects the natural sequence variation seen across the FMDV serotypes, are effective inhibitors for alphavbeta6. In contrast, although RGDM-containing peptides were effective for alphavbeta8, RGDR-containing peptides were not. These observations were confirmed by showing that a virus containing an RGDR motif uses alphavbeta8 less efficiently than alphavbeta6 as a receptor for infection. Finally, evidence is presented that shows alphavbeta3 to be a poor receptor for infection by type O FMDV. Taken together, our data suggest that the integrin binding loop of FMDV has most likely evolved for binding to alphavbeta6 with a higher affinity than to alphavbeta3 and alphavbeta8.     

Use of phage display to probe the evolution of binding specificity and affinity in integrins

The specific binding of RGD-containing proteins to integrin is a function of both the conformation of and the local sequence surrounding the RGD motif. To study the effect of these factors on integrin binding affinity and specificity, we obtained RGD-containing ligands specific for different integrins presented on the same protein scaffold. The beta-turn region between two anti-parallel beta-strands on the loop I of tendamistat, an inhibitor of alpha-amylase, was extended by two residues and randomized in a phagemid library. This library and two subsequently constructed RGD-containing loop I libraries were biopanned with purified integrins alphaIIbbeta3, alphaVbeta3 and alphaVbeta5 individually. The sequence analysis of selected tendamistat variants and characterization by phage ELISA revealed that phage adhesion is mediated exclusively by an RGD motif located at only two out of four possible positions on loop I. Further, sequences flanking the RGD motif were specific for different integrin targets. Interestingly, selected tendamistat variants mimic natural integrin ligands, both in sequence similarity and in integrin binding specificity, indicating that various ligand specificity patterns can be generated by driving towards maximum affinity in the integrin-ligand complexes.     

Decorsin. A potent glycoprotein IIb-IIIa antagonist and platelet aggregation inhibitor from the leech Macrobdella decora

The discovery, purification, and characterization of decorsin, a protein isolated from the North American leech Macrobdella decora, are described. Decorsin acts as an antagonist of platelet glycoprotein IIb-IIIa (GPIIb-IIIa), and is a potent inhibitor of platelet aggregation. The protein was purified to apparent homogeneity from crude whole leech extracts by treatment with trifluoroacetic acid followed by GPIIb-IIIa affinity chromatography and C18 reverse-phase high performance liquid chromatography. Decorsin was also isolated from a solution of leech ingestate by treatment with trifluoroacetic acid followed by C18 reverse-phase high performance liquid chromatography. The primary sequence of decorsin indicates that the protein is 39 amino acids long and contains 6 cysteine and 6 proline residues, as well as the sequence Arg-Gly-Asp, (RGD), a proposed recognition site of many adhesion proteins. A molecular mass of 4379 was obtained by fast atom bombardment mass spectrometry and is consistent with the mass calculated from the observed sequence. Evidence for an N-3 isoform, lacking the first 3 amino-terminal residues is also presented. Both decorsin and the N-3 isoform inhibit GP IIb-IIIa binding to immobilized fibrinogen with an IC50 of approximately 1.5 nM. Human platelet aggregation induced by ADP is inhibited by decorsin with an IC50 of approximately 500 nM; complete inhibition was observed at less than or equal to 1 microM. Based on overall sequence homology, decorsin does not belong to the family of GPIIb-IIIa protein antagonists that is found in snake venoms (Dennis, M. S., Henzel, W. J., Pitti, R. M., Lipari, M. T., Napier, M. A., Deisher, T. A., Bunting, S., and Lazarus, R. A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2471-2475); however the carboxyl-terminal RGD-containing region from residues 27 to 38 of decorsin is approximately 60% homologous with the corresponding region of the snake venom proteins, suggesting that high affinity binding of these proteins to GPIIb-IIIa is defined by this epitope.     

Crystal structure of trimestatin, a disintegrin containing a cell adhesion recognition motif RGD

Disintegrins are a family of small proteins containing an Arg-Gly-Asp (RGD) sequence motif that binds specifically to integrin receptors. Since the integrin is known to serve as the final common pathway leading to aggregation via formation of platelet-platelet bridges, disintegrins act as fibrinogen receptor antagonists. Here, we report the first crystal structure of a disintegrin, trimestatin, found in snake venom. The structure of trimestatin at 1.7A resolution reveals that a number of turns and loops form a rigid core stabilized by six disulfide bonds. Electron densities of the RGD sequence are visible clearly at the tip of a hairpin loop, in such a manner that the Arg and Asp side-chains point in opposite directions. A docking model using the crystal structure of integrin alphaVbeta3 suggests that the Arg binds to the propeller domain, and Asp to the betaA domain. This model indicates that the C-terminal region is another potential binding site with integrin receptors. In addition to the RGD sequence, the structural evidence of a C-terminal region (Arg66, Trp67 and Asn68) important for disintegrin activity allows understanding of the high affinity and selectiveness of snake venom disintegrin for integrin receptors. The crystal structure of trimestatin should provide a useful framework for designing and developing more effective drugs for controlling platelet aggregation and anti-angiogenesis cancer.     

Manganese ion elicits a binding activity of placenta vitronectin receptor to fibronectin cell-binding domain.

Some members of the integrin family recognize the RGD sequence which is common to cell adhesive proteins in a divalent cation-dependent manner. In the presence of Ca2+ and Mg2+, the fibronectin receptor of placenta recognizes the RGD sequence of fibronectin, but not that of vitronectin, while the vitronectin receptor of placenta recognizes the RGD sequence of vitronectin, but not that of fibronectin, although both receptors recognize the same RGD sequence. We have found by performing an enzyme-linked immunosorbent assay (ELISA) using receptor-specific monoclonal antibodies and by electrophoretic analysis that in the presence of Mn2+ a vitronectin receptor of placenta binds to an affinity column coupled with the cell-binding domain of fibronectin. By replacing divalent cations from Mn2+ to Ca2+ and Mg2+, the vitronectin receptor was completely eluted from the column. When the synthetic peptides GRGDSP and GRGESP were applied to the column as competitors, the Mn(2+)-dependent binding was inhibited by both peptides. These results suggest that Mn2+ elicits a binding activity of the placenta vitronectin receptor to the RGD site of fibronectin. The modulation of ligand specificity by Mn2+ will provide an important clue in the elucidation of the cause of individual ligand specificity of RGD-recognizing integrins.     

Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library

Our previous studies showed that the alpha 5 beta 1 integrin selects cysteine pair-containing RGD peptides from a phage display library based on a random hexapeptide. We have therefore searched for more selective peptides for this integrin using a larger phage display library, where heptapeptides are flanked by cysteine residues, thus making the inserts potentially cyclic. Most of the phage sequences that bound to alpha 5 beta 1 (69 of 125) contained the RGD motif. Some of the heptapeptides contained an NGR motif. As the NGR sequence occurs in the cell-binding region of the fibronectin molecule, this sequence could contribute to the specific recognition of fibronectin by alpha 5 beta 1. Selection for high affinity peptides for alpha 5 beta 1 surprisingly yielded a sequence RRETAWA that does not bear obvious resemblance to known integrin ligand sequences. The synthetic cyclic peptide GACRRETAWACGA (*CRRETAWAC*) was a potent inhibitor of alpha 5 beta 1-mediated cell attachment to fibronectin. This peptide is nearly specific for the alpha 5 beta 1 integrin, because much higher concentrations were needed to inhibit the alpha v beta 1 integrin, and there was no effect on alpha v beta 3- and alpha v beta 5-mediated cell attachment to vitronectin. The peptide also did not bind to the alpha IIb beta 3 integrin. *CRRETAWAC* appears to interact with the same or an overlapping binding site in alpha 5 beta 1 as RGD, because cell attachment to *CRRETAWAC* coated on plastic was divalent cation dependent and could be blocked by an RGD-containing peptide. These results reveal a novel binding specificity in the alpha 5 beta 1 integrin.     

Alpha(v)beta3 and alpha(v)beta5 integrins bind both the proximal RGD site and non-RGD motifs within noncollagenous (NC1) domain of the alpha3 chain of type IV collagen: implication for the mechanism of endothelia cell adhesion

The NC1 domains of human type IV collagen, in particular alpha3NC1, are inhibitors of angiogenesis and tumor growth (Petitclerc, E., Boutaud, A., Prestayko, A., Xu, J., Sado, Y., Ninomiya, Y., Sarras, M. P., Jr., Hudson, B. G., and Brooks, P. C. (2000) J. Biol. Chem. 275, 8051-8061). The recombinant alpha3NC1 domain contained a RGD site as part of a short collagenous sequence at the N terminus, designated herein as RGD-alpha3NC1. Others, using synthetic peptides, have concluded that this RGD site is nonfunctional in cell adhesion, and therefore, the anti-angiogenic activity is attributed exclusively to alpha(v)beta(3) integrin interactions with non-RGD motifs of the RGD-alpha3NC1 domain (Maeshima, Y., Colorado, P. C., and Kalluri, R. (2000) J. Biol. Chem. 275, 23745-23750). This nonfunctionality is surprising given that RGD is a binding site for alpha(v)beta(3) integrin in several proteins. In the present study, we used the alpha3NC1 domain with or without the RGD site, expressed in HEK 293 cells for native conformation, as an alternative approach to synthetic peptides to assess the functionality of the RGD site and non-RGD motifs. Our results demonstrate a predominant role of the RGD site for endothelial adhesion and for binding of alpha(v)beta(3) and alpha(v)beta(5) integrins. Moreover, we demonstrate that the two non-RGD peptides, previously identified as the alpha(v)beta(3) integrin-binding sites of the alpha3NC1 domain, are 10-fold less potent in competing for integrin binding than the native protein, indicating the importance of additional structural and/or conformational features of the alpha3NC1 domain for integrin binding. Therefore, the RGD site, in addition to non-RGD motifs, may contribute to the mechanisms of endothelial cell adhesion in the human vasculature and the anti-angiogenic activity of the RGD-alpha3NC1 domain.     

Integrin alpha IIb beta 3 (platelet GPIIb-IIIa) recognizes multiple sites in fibronectin.

The binding of fibronectin (Fn) to several integrins involves the Arg-Gly-Asp (RGD) tripeptide sequence. However, linear synthetic RGD peptides do not completely mimic the cell attachment activity of intact Fn or certain large Fn fragments. This suggests that the integrin-Fn interaction involves a more extended surface of Fn than that provided by the RGD sequence. To test this possibility, three novel monoclonal anti-Fn antibodies that inhibit its binding to a purified integrin, alpha IIb beta 3, were developed. The epitopes of these three antibodies mapped to a region at least 55 residues amino-terminal of the RGD sequence. Further, recombinant fragments of Fn containing these epitopes and lacking the RGD site also inhibited the binding of Fn to purified alpha IIb beta 3. These fragments, which spanned Fn residues 1359-1436, bound to alpha IIb beta 3 in a divalent cation-dependent manner. In addition, this region of Fn bound specifically to alpha IIb beta 3 on thrombin-stimulated but not resting platelets. These results demonstrate the presence of additional sequences in Fn that interact with integrin alpha IIb beta 3 and suggest that multiple sites in Fn are involved in its recognition by this integrin.     

Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly

Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.     

Analysis of a foot-and-mouth disease virus type A24 isolate containing an SGD receptor recognition site in vitro and its pathogenesis in cattle

Foot-and-mouth disease virus (FMDV) initiates infection by binding to integrin receptors via an Arg-Gly-Asp (RGD) sequence found in the G-H loop of the structural protein VP1. Following serial passages of a type A(24) Cruzeiro virus (A(24)Cru) in bovine, via tongue inoculation, a virus was generated which contained an SGD sequence in the cell receptor-binding site and expressed a turbid plaque phenotype in BHK-21 cells. Propagation of this virus in these cells resulted in the rapid selection of viruses that grew to higher titers, produced clear plaques, and now contained an RGD sequence in place of the original SGD. To study the role of the SGD sequence in FMDV receptor recognition and bovine virulence, we assembled an infectious cDNA clone of an RGD-containing A(24)Cru and derived mutant clones containing either SGD with a single nucleotide substitution in the R(144) codon or double substitutions at this position to prevent mutation of the S to an R. The SGD viruses grew poorly in BHK-21 cells and stably maintained the sequence during propagation in BHK-21 cells expressing the bovine alpha(V)beta(6) integrin (BHK3-alpha(V)beta(6)), as well as in experimentally infected and contact steers. While all the SGD-containing viruses used only the bovine alpha(V)beta(6) integrin as a cellular receptor with relatively high efficiency, the revertant RGD viruses utilized either the alpha(V)beta(1) or alpha(V)beta(3) bovine integrins with higher efficiency than alpha(V)beta(6) and grew well in BHK-21 cells. Replacing the R at the -1 SGD position with either K or E showed that this residue did not contribute to integrin utilization in vitro. These results illustrate the rapid evolution of FMDV with alteration in receptor specificity and suggest that viruses with sequences other than RGD, but closely related to it, can still infect via integrin receptors and induce and transmit the disease to susceptible animals.     

Construction and characterization of trifunctional single-chain urokinase-type plasminogen activators.

Two chimeric proteins have been constructed. One consists of four parts: a portion of the low molecular mass single-chain urokinase-type plasminogen activator (scu-PA-32K, residues 144-411), a 15-mer linker sequence, the C-terminal amino-acid sequence (residues 53-65) of hirudin (Hir), and an RGD sequence derived from the leech protein decorsin, i.e. scu-PA(32 k)-linker-Hir (residues 53-65)-RGD peptide. The other comprises two main segments: scu-PA(32 k) and hirudin into which RGDSP is inserted between its residues 33 and 34, i.e. hirudin (residues 1-33)-RGDSP-hirudin (residues 34-65)-scu-PA(32 k). These two chimeric genes were expressed in Escherichia coli, and the products were purified by Zn2+-chelating Sepharose 4B chromatography and benzamidine Sepharose 6B chromatography. Our results suggested that these two chimeric proteins not only had plasminogen-dependent fibrinolytic activity, but also possessed platelet aggregation inhibitory activity and antithrombin activity.     

Amino acid sequence and homology modeling of obtustatin,a novel non-RGD-containing short disintegrin isolated from the venom of Vipera lebetina obtusa

Disintegrins represent a group of cysteine-rich peptides occurring in Crotalidae and Viperidae snake venoms, and are potent antagonists of several integrin receptors. A novel disintegrin, obtustatin, was isolated from the venom of the Vipera lebetina obtusa viper, and represents the first potent and selective inhibitor of the binding of integrin alpha(1)beta(1) to collagen IV. The primary structure of obtustatin contains 41 amino acids and is the shortest disintegrin described to date. Obtustatin shares the pattern of cysteines of other short disintegrins. However, in contrast to known short disintegrins, the integrin-binding loop of obtustatin is two residues shorter and does not express the classical RGD sequence. Using synthetic peptides, a KTS motif was identified as the integrin-binding sequence. A three-dimensional model of obtustatin, built by homology-modeling structure calculations using different templates and alignments, strongly indicates that the novel KTS motif may reside at the tip of a flexible loop.     

Characterization of the type 3 fimbriae with different MrkD adhesins: possible role of the MrkD containing an RGD motif

Four novel mrkD alleles namely mrkD(V1), mrkD(V2), mrkD(V3), and mrkD(V4) were identified in seventeen Klebsiella pneumoniae meningitis strains using PCR-RFLP and sequence determination. Comparative analysis revealed a most variable region containing an RGD motif in the receptor domain of MrkD(V3). In order to determine if the sequence confers the K. pneumoniae mrkD(V3) the highest level of the fimbrial activity, a type 3 fimbriae display system was constructed in Escherichia coli. The E. coli JM109[pmrkABCD(V3)F] displaying meshwork-like fimbriae also had the most fimbrial activity, supporting a possible role of the varied sequences. In a dose-dependent manner, the GRGDSP hexapeptide appeared to inhibit the adhesion of the E. coli JM109[pmrkABCD(V3)F] to HCT-8, an ileocecal epithelial cell line. In addition, the adhesion activity was reduced by the addition of anti-alpha5beta1 integrin monoclonal antibody, indicating that the RGD containing region in MrkD(V3) is responsible for the binding of type 3 fimbriae to integrin.     

Identification of key functional amino acids of the mouse fertilin beta (ADAM2) disintegrin loop for cell-cell adhesion during fertilization

Fertilin beta (also known as ADAM2) is a cell adhesion molecule on the surface of mammalian sperm that participates in sperm-egg membrane binding. Fertilin beta is a member of the molecular family known as ADAMs or MDCs. These proteins have a disintegrin domain with homology to integrin ligands found in snake venoms; several of these snake proteins have an RGD tripeptide presented on an extended "disintegrin loop." However, fertilin beta lacks an RGD tripeptide and instead has the consensus sequence X(D/E)ECD (QDECD in mouse fertilin beta) in its putative disintegrin loop, and there is controversy over which amino acids comprise the active site of the fertilin beta disintegrin loop. We have used point-mutated versions of the sequence AQDECDVT and two bioassays to identify the key functional amino acids of this sequence from the mouse fertilin beta disintegrin domain. Amino acid substitutions for the terminal aspartic acid residue of the QDECD sequence result in dramatically reduced activities in the two assays for protein function, implicating the terminal aspartic acid residue as critical for protein function. Substitutions for the glutamic acid and the cysteine residues in the QDECD sequence result in slight reductions in activity, whereas substitution of the first aspartic acid has virtually no effect. These data suggest that the conserved ECD sequence of the mouse fertilin beta disintegrin loop, especially the terminal D residue, contributes more to the protein's activity than does the QDE sequence that aligns with the RGD tripeptide in other disintegrins.