Polydom/SVEP1 Is a Ligand for Integrin α9β1

This research was originally published in the Journal of Biological Chemistry. Ryoko Sato-Nishiuchi, Itsuko Nakano, Akio Ozawa, Yuya Sato, Makiko Takeichi, Daiji Kiyozumi, Kiyoshi Yamazaki, Teruo Yasunaga, Sugiko Futaki and Kiyotoshi Sekiguchi. Polydom/SVEP1 Is a Ligand for Integrin α9β1. J. Biol. Chem. 2012; 287: 25615-25630 © the American Society for Biochemistry and Molecular Biolog

Laminin Isoforms Containing the γ3 Chain Are Unable to Bind to Integrins due to the Absence of the Glutamic Acid Residue Conserved in the C-terminal Regions of the γ1 and γ2 Chains*S⃞

Laminins are the major cell adhesive proteins in basement membranes, and consist of three subunits termed α, β, and γ. Recently, we found that the Glu residue at the third position from the C termini of the γ1 and γ2 chains is critically involved in integrin binding by laminins. However, the γ3 chain lacks this Glu residue, suggesting that laminin isoforms containing the γ3 chain may be unable to bind to integrins. To address this possibility, we expressed the E8 fragment of laminin-213 and found that it was incapable of binding to integrins. Similarly, the E8 fragment of laminin-113 was expressed and also found to be inactive in binding to integrins, confirming the distinction between the integrin binding activities of γ3 chain-containing isoforms and those containing the γ1 or γ2 chain. To further address the importance of the Glu residue, we swapped the C-terminal four amino acids of the γ3 chain with the C-terminal nine amino acids of the γ1 chain, which contain the Glu residue. The resulting chimeric E8 fragment of laminin-213 became fully active in integrin binding, whereas replacement with the nine amino acids of the γ1 chain after substitution of Gln for the conserved Glu residue failed to restore the integrin binding activity. These results provide both loss-of-function and gain-of-function evidence that laminin isoforms containing the γ3 chain are unable to bind to integrins due to the absence of the conserved Glu residue, which should play a critical role in integrin binding by laminins

Probing the interaction of tetraspanin CD151 with integrin α3β1 using a panel of monoclonal antibodies with distinct reactivities toward the CD151-integrin α3β1 complex

CD151, a member of the tetraspanin family of proteins, forms a stable complex with integrin α3β1 and regulates integrin-mediated cell-substrate adhesion. However, the molecular basis of the stable association of CD151 with integrin α3β1 remains poorly understood. In the present study, we show that a panel of anti-human CD151 mAbs (monoclonal antibodies) could be divided into three groups on the basis of their abilities to co-immunoprecipitate integrin α3: Group-1 mAbs were devoid of sufficient activities to co-precipitate integrin α3 under both low- and high-stringency detergent conditions; Group-2 mAbs co-precipitated integrin α3 under low-stringency conditions; and Group-3 mAbs exhibited strong co-precipitating activities under both conditions. Group-1 mAbs in particular exhibited increased reactivity toward integrin α3β1-unbound CD151, indicating that the binding sites for Group-1 mAbs are partly blocked by bound integrin α3β1. Epitope mapping using a series of CD151 mutants with substitutions at amino acid residues that are not conserved between human and mouse CD151 revealed that Gly¹⁷⁶/Gly¹⁷⁷, Leu¹⁹¹ and Gln¹⁹⁴ comprise epitopes characteristic of Group-1 mAbs. Replacement of short peptide segments, each containing one of these epitopes, with those of other tetraspanins lacking stable interactions with integrin α3β1 demonstrated that the segment from Cys¹⁸⁵ to Cys¹⁹², including Leu¹⁹¹, was involved in the stable association of CD151 with integrin α3β1, as was the Gln¹⁹⁴-containing QRD peptide. Taken together these results indicate that two consecutive segments including two Group-1 epitopes, Leu¹⁹¹ and Gln¹⁹⁴, comprise an interface between CD151 and integrin α3β1, and, along with the epitope including Gly¹⁷⁶/Gly¹⁷⁷, are concealed by bound integrin

The C-terminal Region of Laminin β Chains Modulates the Integrin Binding Affinities of Laminins*S⃞

Laminins are major cell-adhesive proteins in basement membranes that are capable of binding to integrins. Laminins consist of three chains (α, β, and γ), in which three laminin globular modules in the α chain and the Glu residue in the C-terminal tail of the γ chain have been shown to be prerequisites for binding to integrins. However, it remains unknown whether any part of the β chain is involved in laminin-integrin interactions. We compared the binding affinities of pairs of laminin isoforms containing the β1 or β2 chain toward a panel of laminin-binding integrins, and we found that β2 chain-containing laminins (β2-laminins) bound more avidly to α3β1 and α7X2β1 integrins than β1 chain-containing laminins (β1-laminins), whereas α6β1, α6β4, and α7X1β1 integrins did not show any preference toward β2-laminins. Because α3β1 contains the “X2-type” variable region in the α3 subunit and α6β1 and α6β4 contain the “X1-type” region in the α6 subunit, we hypothesized that only integrins containing the X2-type region were capable of discriminating between β1-laminins and β2-laminins. In support of this possibility, a putative X2-type variant of α6β1 was produced and found to bind preferentially to β2-laminins. Production of a series of swap mutants between the β1 and β2 chains revealed that the C-terminal 20 amino acids in the coiled-coil domain were responsible for the enhanced integrin binding by β2-laminins. Taken together, the results provide evidence that the C-terminal region of β chains is involved in laminin recognition by integrins and modulates the binding affinities of laminins toward X2-type integrins

Virological characteristics correlating with SARS-CoV-2 spike protein fusogenicity

IntroductionThe severe acute respiratory syndrome coronavirus (SARS-CoV-2) spike (S) protein is essential in mediating membrane fusion of the virus with the target cells. Several reports demonstrated that SARS-CoV-2 S protein fusogenicity is reportedly closely associated with the intrinsic pathogenicity of the virus determined using hamster models. However, the association between S protein fusogenicity and other virological parameters remains elusive.MethodsIn this study, we investigated the virological parameters (e.g., S1/S2 cleavage efficiency, plaque size, pseudoviral infectivity, pseudovirus entry efficiency, and viral replication kinetics) of eleven previous variants of concern (VOCs) and variants of interest (VOIs) correlating with S protein fusogenicity.Results and discussionS protein fusogenicity was found to be strongly correlated with S1/S2 cleavage efficiency and plaque size formed by clinical isolates. However, S protein fusogenicity was less associated with pseudoviral infectivity, pseudovirus entry efficiency, and viral replication kinetics. Taken together, our results suggest that S1/S2 cleavage efficiency and plaque size could be potential indicators to predict the intrinsic pathogenicity and S protein fusogenicity of newly emerged SARS-CoV-2 variants