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• W2022026424 abstract "The ability of laminins to self-polymerize is crucial for the formation of basement membranes. Development of this polymerized network has profound effects upon tissue architecture as well as on the intracellular organization and differentiation of neighboring cells. The laminin N-terminal (LN) domains have been shown to mediate this interaction and studies using proteolytic fragments derived from laminin-1 led to the theory that network assembly depends on the formation of a heterotrimeric complex between LN domains derived from α, β, and γ chains in different laminin molecules with homologous interactions being insignificant. The laminin family consists of 15 known isoforms formed from five α, three β, and three γ chains, of which some are truncated and lack the N-terminal LN domain. To address whether the model of heterotrimeric complex formation is applicable to laminin isoforms other than laminin-1, eight LN domains found in the laminin protein family were recombinantly expressed and tested in three different assays for homologous and heterologous interactions. The results showed that the lack of homologous interactions is an exception, with such interactions being seen for LN domains derived from all α chains and from the β and2 β3 subunits. The γ chain-derived LN domains showed a far more limited binding repertoire, particularly in the case of the γ3 chain, which is found present in a range of non-basement membrane locations. Further, whereas the interactions depended upon Ca2+ ions, with EDTA reversibly abrogating binding, EDTA-induced conformational changes were not reversible. Together these results demonstrate that the assembly model proposed on the basis of laminin-1 may be a simplification, with the assembly of naturally occurring laminin networks being far more complex and highly dependent upon which laminin isoforms are present. The ability of laminins to self-polymerize is crucial for the formation of basement membranes. Development of this polymerized network has profound effects upon tissue architecture as well as on the intracellular organization and differentiation of neighboring cells. The laminin N-terminal (LN) domains have been shown to mediate this interaction and studies using proteolytic fragments derived from laminin-1 led to the theory that network assembly depends on the formation of a heterotrimeric complex between LN domains derived from α, β, and γ chains in different laminin molecules with homologous interactions being insignificant. The laminin family consists of 15 known isoforms formed from five α, three β, and three γ chains, of which some are truncated and lack the N-terminal LN domain. To address whether the model of heterotrimeric complex formation is applicable to laminin isoforms other than laminin-1, eight LN domains found in the laminin protein family were recombinantly expressed and tested in three different assays for homologous and heterologous interactions. The results showed that the lack of homologous interactions is an exception, with such interactions being seen for LN domains derived from all α chains and from the β and2 β3 subunits. The γ chain-derived LN domains showed a far more limited binding repertoire, particularly in the case of the γ3 chain, which is found present in a range of non-basement membrane locations. Further, whereas the interactions depended upon Ca2+ ions, with EDTA reversibly abrogating binding, EDTA-induced conformational changes were not reversible. Together these results demonstrate that the assembly model proposed on the basis of laminin-1 may be a simplification, with the assembly of naturally occurring laminin networks being far more complex and highly dependent upon which laminin isoforms are present. Basement membranes are specialized extracellular matrices found underlying all epithelia and endothelia as well as surrounding many types of mesenchymal cells. Laminins constitute the major noncollagenous protein component within the basement membrane and are crucial for its formation (1De Arcangelis A. Neuville P. Boukamel R. Lefebvre O. Kedinger M. Simon-Assmann P. J. Cell Biol. 1996; 133: 417-430Crossref PubMed Scopus (133) Google Scholar, 2Smyth N. Vatansever H.S. Murray P. Meyer M. Frie C. Paulsson M. Edgar D. J. Cell Biol. 1999; 144: 151-160Crossref PubMed Scopus (427) Google Scholar). Through their interactions with specific receptors, especially members of the β1 integrin family and α-dystroglycan, they induce many cellular effects, including differentiation and cellular and axonal migration. The prototype, laminin-1, isolated from embryonic carcinoma cells (3Chung A.E. Jaffe R. Freeman I.L. Vergnes J.-P. Braginski J.E. Carlin B. Cell. 1979; 16: 277-287Abstract Full Text PDF PubMed Scopus (241) Google Scholar) or the Engelbreth-Holm-Swarm tumor (4Timpl R. Rohde H. Gehron Robey P. Rennard S.I. Foidart J.-M. Martin G.R. J. Biol. Chem. 1979; 254: 9933-9937Abstract Full Text PDF PubMed Google Scholar) has been shown to belong to a family consisting of 15 members (for nomenclature, see Refs. 5Burgeson R.E. Chiquet M. Deutzmann R. Ekblom P. Engel J. Kleinman H. Martin G.R. Meneguzzi G. Paulsson M. Sanes J. Timpl R. Tryggvason K. Yamada Y. Yurchenco P.D. Matrix Biol. 1994; 14: 209-211Crossref PubMed Scopus (696) Google Scholar, 6Tunggal P. Smyth N. Paulsson M. Ott M.C. Microsc. Res. Tech. 2000; 51: 214-227Crossref PubMed Scopus (168) Google Scholar, 7Libby R.T. Champliaud M.F. Claudepierre T. Xu Y. Gibbons E.P. Koch M. Burgeson R.E. Hunter D.D. Brunken W.J. J. Neurosci. 2000; 20: 6517-6528Crossref PubMed Google Scholar). Laminins are multidomain heterotrimers formed by the combination of one α, one β, and one γ chain. Laminin-1 is an 800-kDa glycoprotein composed of a 400-kDa α1 chain and β1 and γ1 chains, each of 200 kDa. It has a cross structure with one long and three short arms, the latter being formed from the three free N-terminal ends of the α, β, and γ chains (8Engel J. Odermatt E. Engel A. Madri J.A. Furthmayr H. Rohde H. Timpl R. J. Mol. Biol. 1981; 150: 97-120Crossref PubMed Scopus (476) Google Scholar). These parts of the β and γ chains each contain two globular domains, designated IV and VI, whereas there are three, IVa, IVb, and VI, in the short arm contributed by the α chain. The globules are interspersed by multiple laminin epidermal growth factor-like (LE) 1The abbreviations used are: LE, laminin epidermal growth factor-like; LN, laminin N-terminal; xMAP, flexible multianalyte profiling; Bicine, N,N-bis(2-hydroxyethyl)glycine; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; BS3, bis(sulfosuccinimidyl)suberate. domains forming rods. The VI domains are also designated laminin N-terminal (LN) domains, and this nomenclature will be used throughout. As well as being found in full-length laminin chains, LN domains are also present in netrins, secreted molecules acting as guidance cues in neuronal path finding. The α1, β1, and γ1 subunits have been shown to be representatives of three distinct but closely related gene families, which consist of five, three, and three known members, respectively. Certain chains have been described as maintaining the domain structure of those in the original laminin-1 (e.g. α2 and β2) whereas others have N-terminal truncations presumably acquired in the process of evolutionary duplication (e.g. α4 and γ2) (9Maurer P. Engel J. Ekblom P. Timpl R. The Laminins. Harwood Academic Publishers, Amsterdam1996: 27-50Google Scholar). The trimeric molecules formed by the combination of these chains may appear to have no short arms (e.g. laminin-5) (α3β3γ2) (10Rousselle P. Lunstrum G.P. Keene D.R. Burgeson R.E. J. Cell Biol. 1991; 114: 567-576Crossref PubMed Scopus (658) Google Scholar) or have a Y shape on electron microscopy (e.g. laminin-7) (α3β2γ1) (11Champliaud M.F. Lunstrum G.P. Rousselle P. Nishiyama T. Keene D.R. Burgeson R.E. J. Cell Biol. 1996; 132: 1189-1198Crossref PubMed Scopus (228) Google Scholar). The laminin trimer is formed by assembly of the coiled-coil α-helical regions of the three chains, which results in the formation of the long arm structure (12Paulsson M. Deutzmann R. Timpl R. Dalzoppo D. Odermatt E. Engel J. EMBO J. 1985; 4: 309-316Crossref PubMed Scopus (137) Google Scholar). The amino acid sequence in these regions must contain additional information, beyond the basic heptad repeat with hydrophobic residues in positions 1 and 4, since many but not all the theoretically possible heterotrimers are observed in vivo. Possibly, partially exclusive ionic bridges between the different chains play a role in increasing the stability in the formation of the coiled-coil α-helix (13Beck K. Dixon T.W. Engel J. Parry D.A.D. J. Mol. Biol. 1992; 231: 311-323Crossref Scopus (84) Google Scholar). Upon secretion, the laminin molecules aggregate to form a meshwork (14Yurchenco P.D. Tsilibary E.C. Charonis A.S. Furthmayr H. J. Biol. Chem. 1985; 260: 7636-7644Abstract Full Text PDF PubMed Google Scholar). In vitro this self-assembly is dependant upon Ca2+ (15Paulsson M. J. Biol. Chem. 1988; 263: 5425-5430Abstract Full Text PDF PubMed Google Scholar) and is inhibited by chelating agents (14Yurchenco P.D. Tsilibary E.C. Charonis A.S. Furthmayr H. J. Biol. Chem. 1985; 260: 7636-7644Abstract Full Text PDF PubMed Google Scholar). At high concentrations and in the presence of Ca2+, the laminin molecules align to produce an array through the interaction of the N-terminal globular LN domains (16Yurchenco P.D. Cheng Y.S. Colgnato H. J. Cell Biol. 1992; 117: 1119-1133Crossref PubMed Scopus (225) Google Scholar). Calcium ions are believed to produce a conformational change in the LN domains, allowing aggregation (15Paulsson M. J. Biol. Chem. 1988; 263: 5425-5430Abstract Full Text PDF PubMed Google Scholar, 17Paulsson M. Saladin K. J. Biol. Chem. 1989; 264: 18726-18732Abstract Full Text PDF PubMed Google Scholar). The self-aggregation of laminin-1 is shared by many “full-sized” laminin isoforms but not by laminin-5 and -6, which contain chains with N-terminal truncations and thereby lack one or more LN domains (18Cheng Y.S. Champliaud M.F. Burgeson R.E. Marinkovich M.P. Yurchenco P.D. J. Biol. Chem. 1997; 272: 31525-31532Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). The polymerization of laminin-1 may be inhibited by proteolytic fragments that contain LN domains (19Schittny J.C. Yurchenco P.D. J. Cell Biol. 1990; 110: 825-832Crossref PubMed Scopus (90) Google Scholar, 20Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar). Fragment E4, containing the LN domain of the β1 chain, binds to a fragment representing the LN domains of the other two laminin-1 short arms with a KD of 1.4 μm, whereas homologous self-interactions are either very weak or do not occur (20Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar). More recently, recombinant fragments representing the short arms of the α chains have also been shown able to inhibit laminin-1 polymer formation (21Colognato-Pyke H. O'Rear J.J. Yamada Y. Carbonetto S. Cheng Y.S. Yurchenco P.D. J. Biol. Chem. 1995; 270: 9398-9406Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 22Garbe J.H. Gohring W. Mann K. Timpl R. Sasaki T. Biochem. J. 2002; 362: 213-221Crossref PubMed Scopus (55) Google Scholar). Laminin self-aggregation is not the only mechanism needed for correct basement membrane assembly, since self-aggregation preferentially occurs while laminin is bound to cell surface receptors such as α-dystroglycan or integrins (23Cohen M.W. Jacobson C. Yurchenco P.D. Morris G.E. Carbonetto S. J. Cell Biol. 1997; 136: 1047-1058Crossref PubMed Scopus (71) Google Scholar, 24Colognato H. Yurchenco P.D. Curr. Biol. 1999; 18: 1327-1330Abstract Full Text Full Text PDF Google Scholar). However, upon binding to the laminin network, these receptors are also redistributed, which leads to a reorganization of the cortical cytoskeleton (25Colognato H. Winkelmann D.A. Yurchenco P.D. J. Cell Biol. 1999; 145: 619-631Crossref PubMed Scopus (255) Google Scholar). Thus, the aggregation state of laminin in the basement membrane may have regulatory effects on cells. This is highlighted in organ culture experiments, where polymerization-blocking antibodies against laminin or laminin short arm structures inhibited basement membrane assembly and epithelial cell polarization (26Schuger L. Yurchenco P. Relan N.K. Yang Y. Int. J. Dev. Biol. 1998; 42: 217-220PubMed Google Scholar) and smooth muscle cell differentiation (27Yang Y. Palmer K.C. Relan N. Diglio C. Schuger L. Development. 1998; 125: 2621-2629PubMed Google Scholar). Recent studies have also shown that polymerization-deficient laminins fail not only to form a basement membrane-like structure but are also unable to induce differentiation in embryonic stem cells (28Li S. Harrison D. Carbonetto S. Fassler R. Smyth N. Edgar D. Yurchenco P.D. J. Cell Biol. 2002; 157: 1279-1290Crossref PubMed Scopus (256) Google Scholar). Together these studies suggest that regulated laminin aggregation is highly significant for basement membrane-induced cellular differentiation and organogenesis. To understand the self-interaction mechanism of the laminin molecules in more detail, we have expressed recombinantly the LN domains from eight of the mouse laminin chains and have used these in binding assays. We could show that Ca2+ binding was shared between all of the chains but also that they have differing affinity for one another. This suggests that different laminin trimers may have a highly variable arrangement within the basement membrane. Expression and Purification of Murine LN Domains—Total RNA from adult mouse kidney was used as template for reverse transcription in all cases with the exception of β3 and γ3 subunits where RNA of skin from 3-day-old mice was chosen. Reverse transcription followed by PCR was carried out using the primers shown (Supplementary Table I). The amplified DNA fragments were digested with SpeI and NotI and cloned into the NheI-NotI-digested expression vector pCEP-pu BM40-cHis or pCEP-puBM40-cStrep (29Smyth N. Odenthal U. Merkl B. Paulsson M. Methods Mol. Biol. 1999; 139: 49-57Google Scholar). This produces a fusion protein where either a His6 tag or a Strep II tag is placed in frame with the LN coding regions. In the case of the γ1 domain, which contains an internal NotI site, the cDNA was cloned SpeI-XhoI into the same vectors. The plasmids were transfected into 293-EBNA cells by electroporation, and the cells were subsequently selected for puromycin resistance. Serum-free supernatants were tested for expression, after SDS-PAGE separation on a 10% gel, by immunoblotting using antibodies specific for either the His6 tag (penta-His horseradish peroxidase-conjugated mouse monoclonal antibody, Qiagen, Germany) or the Strep II tag (rabbit polyclonal serum; IBA, Germany). For His6 tag purification, supernatants were loaded on an IMAC column (Talon metal affinity resin; Clontech) with a flow rate of 0.5 ml/min. After washing with 5 column volumes of buffer (20 mm Hepes, 100 mm NaCl, pH 8.0) containing 2.5 mm imidazol, the proteins were eluted with a linear gradient between 2.5 and 250 mm imidazol. In the case of Strep II-tagged proteins, supernatants were loaded on a StrepTactin-Sepharose column (IBA) at a flow of 0.3 ml/min. After washing with 10 column volumes of 100 mm Tris, pH 8.0, the proteins were eluted with the same buffer containing 2.5 mm desthiobiotin. Native laminin-5, obtained after affinity purification from cell culture media, was a kind gift from Dr. R. Burgeson. Native PAGE—Nondenaturing polyacrylamide gel electrophoresis was performed on a 6% gel in a buffer containing 40 mm Bicine and 15 mm Tris at pH 8.3. Since LNα1 + 2LE and LNβ3 + 4LE did not run toward the anode under these conditions, they were rerun after switching the electrodes. Deglycosylation—Deglycosylation was performed in 20 mm Tris, pH 8.0, or in denaturing buffer (New England Biolabs) after boiling for 10 min. Three μg of protein were incubated overnight with 200 units PNGase F (New England Biolabs) at 37 °C. Circular Dichroism—CD spectra were measured in a Jasco J-715 spectropolarimeter. Proteins were dialyzed against 5 mm Tris, pH 7.5, and had a concentration of 100 μg/ml. Measurements were performed in the presence of 2 mm CaCl2 or after the subsequent addition of EDTA to give a final concentration of 4 mm. Surface Plasmon Resonance Binding Assays—Assays were performed using a Biacore 2000 (BIAcore AB). Coupling of proteins to the CM5 chip was performed in 10 mm sodium acetate, pH 5.0, at a flow rate of 5 μl/min. A 7-min pulse of 0.05 mm N-hydroxysuccinimide, 0.2 m N-ethyl-N′-dimethylaminopropyl carbodiimide was used to activate the surface. The protein was injected until the desired amount was coupled (300–800 response units), and excess reactive groups were deactivated by a 7-min pulse of 1 m ethanolamine hydrochloride, pH 8.5. Measurements were carried out in HBS (20 mm Hepes, 150 mm NaCl, pH 7.4) containing 2 mm CaCl2 or 2 mm EDTA at a flow rate of 30 μl/min. The injection of 120 μl of the protein solution (0.1–10 μm) was followed by a 500-s dissociation. Each analysis was carried out a minimum of three times. The data were analyzed with BIAevaluation software version 3.0 according to the Langmuir model for 1:1 binding. All binding curves could be fitted with an accuracy of χ2 < 0.5 (see Supplementary Tables II–VI). xMAP-Luminex Binding Assays—Measurements were performed in a final total volume of 60 μ l of 10 mm Hepes, 150 mm NaCl, 2 mm CaCl2, pH 7.4. For each data point 1 μl of stock nitrilotriacetic acid-coupled microspheres (Qiagen) was incubated with an excess of His6-tagged ligand for 4 h at 4 °C. After washing the microspheres, the analyte protein was added to give final concentrations from 0–200 μm. Analyte and ligand were gently agitated for 2 h at 25 °C before the addition of penta-His-Alexa 532-conjugated monoclonal antibody (Qiagen). The reaction mixture was then incubated for a further 90 min. Assays with microspheres alone and assays in the absence of the analyte were included as negative controls. Samples were analyzed using a Liqui-Chip-100 work station (Qiagen), and the level of fluorescence at 532 nm was measured. In the absence of analyte, only background signal was observed, indicating no binding of the penta-His antibody to either the microsphere or the coupled ligand. Cross-linking Assays—Cross-linking assays were carried out using the lysine side chain-reactive cross-linker bis(sulfosuccinimidyl)suberate (BS3) (Pierce). The proteins were added at a concentration of 2.5 mm, and the reaction was carried out in a final volume of 40 μl in HBS buffer and in the presence of 2 mm CaCl2. For most assays, the cross-linker was used at a 200-fold molar excess over the protein concentration. The reaction was allowed to continue for 30 min at 25 °C and was stopped by the addition of 5 μl of 0.5 m Tris, pH 7.4. MALDI-TOF Mass Spectrometry—For MALDI-TOF mass spectrometry analysis, the samples were dissolved in 5 μl of 0.1% aqueous trifluoroacetic acid. MALDI mass spectrometry was carried out in linear mode on a Bruker Reflex IV equipped with a video system, a nitrogen UV laser (Omax = 337 nm), and a HiMass detector. 1 μl of the sample solution was placed on the target, and 1 μl of a freshly prepared saturated solution of sinapinic acid in acetonitrile/H2O (2:1) with 0.1% trifluoroacetic acid was added. The spot was then recrystallized by the addition of another 1 μl of acetonitrile/H2O (2:1), which resulted in a fine crystalline matrix. For recording of the spectra, an acceleration voltage of 20 kV was used, and the detector voltage was adjusted to 1.9 kV. Approximately 500 single laser shots were summed into an accumulated spectrum. Calibration was carried out using the single and doubly protonated ion signal of bovine serum albumin for external calibration. Expression and Purification of Laminin LN Domains—LN domains from eight full-length murine laminin chains were expressed and purified. Initial attempts to produce these domains without adjacent LE modules failed; however, recombinant expression did occur upon introducing one or more of the LE modules from domain V. The expression level was dependent upon the number of LE modules following the LN domain and varied between 2 and 15 mg/liter of conditioned medium, with the highest yields obtained from constructs containing four LE domains (Fig. 1). The individual domains were purified from the serum-free medium either by immobilized metal ion affinity chromatography for the His6-tagged forms or by StrepTactin affinity chromatography where the Strep II-tag was used. Single step purification was sufficient to obtain the products at a high degree of purity (Fig. 2a). This was carried out at 4 °C, and protease inhibitors were used in all solutions; however, EDTA was omitted as it could possibly affect later divalent cation-dependent assays. SDS-PAGE of the purified recombinant proteins, without reduction of disulfide bonds, revealed distinct but weaker double bands for the β1- and β2-derived proteins (Fig. 2). These largely resolved upon reduction, which suggested that the proteins possibly had incomplete or variable disulfide closure. Also in the case of the γ3 LN domain, two bands were seen. However, these remained after treatment with β-mercaptoethanol, raising the possibility that a proportion of the protein had undergone limited proteolysis or some other post-translational modification. To investigate this, we tested all of the expressed LN domains for evidence of proteolysis. Edman degradation of each of the proteins revealed the expected N-terminal amino acid sequence (APLV), produced through cleavage by the signal peptidase of the BM40 fusion signal peptide used instead of the native signal sequence. Since the proteins were produced with C-terminal tags, immunoreactivity with tag-specific antibodies showed that C-terminal degradation had not occurred. The LN domains are all potentially N-glycosylated, with the α chains carrying the most asparagine residues suitable for substitution. Deglycosylation with endoglycosidase F resulted in a shift in electrophoretic mobility for all of the proteins, which was most marked in the cases of the α and γ chains (Fig. 2b). For the γ3 fragment, deglycosylation resulted in a single band showing that the two bands of differing mobility were due to variations in N-linked glycosylation. Analysis of the purified proteins by native PAGE in the absence of Ca2+ (Fig. 3) showed the products to run as single bands, suggesting that under these conditions and concentrations self-aggregation was not a major feature. LN Domain Interactions—Three independent assays were carried out to show interactions between the LN domains. Surface plasmon resonance was used to show binding and to obtain ka and kd values. All of the possible interactions were carried out in both directions (i.e. both with the proteins coupled and free in solution). These results were verified using the xMAP suspension binding assays. Finally, certain interactions were confirmed and further studied by covalent cross-linking using the water-soluble cross-linking agent BS3. Since laminin-1 polymerization has been the focus of most previous studies, our initial experiments using surface plasmon resonance tested the α1, β1, and γ1 LN domains found present in laminin-1 (Fig. 4, Table I, and Supplementary Tables II–VI). These assays were carried out at 25 °C, the temperature commonly used for such experiments. However, since temperature has been shown important in LN domain interactions, the analysis was repeated at 13 and 37 °C. At 13 °C, no binding was observed, whereas at 37 °C, signals indicative of aggregation occurred with nonreversible binding being seen. As expected from earlier experiments with proteolytic fragments (20Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar), binding between heterologous laminin-1 LN domains could be detected; a typical response curve is given in Fig. 4, showing the interactions of the α1 and β1 LN domains with a KD of 6.2 × 10–7m. Similarly, binding assays using the recombinant β1 LN domain showed that this failed to self-interact, again in agreement with the previous findings for the E4 fragment, which contains this domain. This lack of self-interaction was also seen for the γ1 LN domain, but the α1 domain interacted with a higher affinity (KD of 1.8 × 10–7m) with itself than seen for any heterotypic interactions of the LN domains of this laminin isoform. This clearly shows that homotypic interactions occur and can influence laminin network assembly.Table IKD values of interacting LN domains as measured by surface plasmon resonanceProtein on chipProtein in solutionα1α2α5β1β2β3γ1γ3α11.80 × 10-75.10 × 10-79.20 × 10-74.20 × 10-71.10 × 10-78.90 × 10-81.80 × 10-6NBaNB, no binding.α23.70 × 10-72.00 × 10-85.50 × 10-75.40 × 10-71.20 × 10-75.20 × 10-8NBNBα51.00 × 10-64.60 × 10-78.05 × 10-81.30 × 10-62.10 × 10-77.40 × 10-86.80 × 10-7NBβ12.40 × 10-86.50 × 10-71.22 × 10-7NB2.80 × 10-84.30 × 10-86.20 × 10-7NBβ29.40 × 10-83.20 × 10-72.00 × 10-73.60 × 10-87.00 × 10-85.30 × 10-82.60 × 10-74.10 × 10-7β38.40 × 10-86.18 × 10-75.50 × 10-75.30 × 10-84.00 × 10-84.90 × 10-81.40 × 10-71.70 × 10-7γ11.20 × 10-7NB5.50 × 10-74.70 × 10-71.70 × 10-71.50 × 10-7NBNBγ3NBNBNBNB1.00 × 10-71.80 × 10-7NBNBa NB, no binding. Open table in a new tab We then studied the possibility of self-interaction for the other laminin LN domains by surface plasmon resonance (Table I and Supplementary Tables II–VI). We found that both the α2 and α5 LN domains self-interact in a manner similar to that of the α1 domain. Whereas the γ3-derived LN domain, like its γ1 counterpart in laminin-1, failed to self-interact, both β2 and β3 LN domains did, in contrast to the results obtained for β1 LN domain. In studies of the interactions between LN domains from the same class of laminin chain, we found evidence of α-α and β-β intragroup heterodimers for all members. However, the two γ chain-derived LN domains showed no interaction. When intergroup binding was analyzed, α-β interactions were seen with all combinations, whereas the γ LN domains again showed a more limited binding repertoire. The γ1 LN domain could be shown to bind to all β chains; however, it failed to interact with the α2 LN domain despite binding to those of α1 and α5. The γ3 LN domain is the most highly restricted in its binding partners, failing to bind to any α chain LN domain, and interacting only with those of β2 and β3. Previous studies have shown the importance of Ca2+ in laminin gel formation. To study this further, the role of divalent cations was evaluated through the addition of EDTA to the analyte in the surface plasmon resonance experiments. In all cases, binding could be inhibited upon Ca2+ removal, a change that was reversible upon the readdition of Ca2+ ions. Circular dichroism spectra were measured and showed that there was a marked reduction in secondary structure for most but not all domains in the absence of Ca2+ ions, since there was no evidence for a change in conformation for the α2-, α5-, or γ3-derived LN domains (Fig. 5). Independent results on the LN domain interactions were obtained using a xMAP suspension assay. Here the ligand is bound in an orientated manner to the surface of a fluorescent microbead via its His6 tag. After washing to remove unbound ligand, the beads are incubated with the analyte. The aligned microbeads are passed in single file through two lasers, and any interaction is detected by measuring a second fluorochrome emitting at a separate wavelength, which is coupled to an antibody directed against this analyte. The degree of antibody fluorescence (i.e. the ratio between the background signal and that occurring as the bead passes the lasers) is obtained to give relative fluorescence response units. In our study, antibodies to the His6 tag were used to detect the interaction. Initial experiments showed that protein binding to the beads resulted in the masking of its His6 tag from the penta-His antibody, thus allowing binding studies to be carried out of two proteins carrying the same tag. To compare the results of the xMAP and the surface plasmon resonance assays, primary studies with various protein combinations were carried out, bringing the analyte concentration to excess. To obtain quantitative results, high levels of the detecting antibody were required as increased free analyte competes for antibody binding to the bead complex. The observed KD values were comparable with those obtained from surface plasmon resonance (Table II). All possible interactions were then analyzed in one direction and showed similar results to those seen in surface plasmon resonance (Table III), with the one exception of the β2-β2 LN domain interaction, where for unknown reasons high background made it impossible to verify a specific interaction. Typical binding results are shown in Fig. 6 for β2 LN domains coupled to the bead and the β3 LN domain in solution.Table IIComparison of KD values obtained by surface plasmon resonance and by xMAP-Luminex analysisLigandAnalyteKD-xMAP[M]KD-SPR[M]aSPR, surface plasmon resonance.β1β1NBbNB, no binding.NBβ1β21.23 × 10-82.80 × 10-8β1β34.94 × 10-84.30 × 10-8β2β33.98 × 10-95.30 × 10-8β3β31.75 × 10-84.90 × 10-8β1γ13.34 × 10-86.20 × 10-7γ1β26.48 × 10-91.70 × 10-7γ1β33.47 × 10-81.50 × 10-7γ3β1NBNBγ3β28.53 × 10-91.00 × 10-7γ3β32.63 × 10-81.80 × 10-7γ1γ1NBNBγ1γ3NBNBγ3γ3NBNBα1β15.43 × 10-84.20 × 10-7a SPR, surface plasmon resonance.b NB, no binding. Open table in a new tab" @default.
• W2022026424 created "2016-06-24" @default.
• W2022026424 creator A5025683370 @default.
• W2022026424 creator A5043042218 @default.
• W2022026424 creator A5049839584 @default.
• W2022026424 creator A5051627035 @default.
• W2022026424 creator A5052471573 @default.
• W2022026424 creator A5061398950 @default.
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• W2022026424 date "2004-10-01" @default.
• W2022026424 modified "2023-09-29" @default.
• W2022026424 title "Molecular Analysis of Laminin N-terminal Domains Mediating Self-interactions" @default.
• W2022026424 cites W1496520030 @default.
• W2022026424 cites W1507170913 @default.
• W2022026424 cites W1523307634 @default.
• W2022026424 cites W1572837727 @default.
• W2022026424 cites W1579619270 @default.
• W2022026424 cites W1585193578 @default.
• W2022026424 cites W1628719417 @default.
• W2022026424 cites W1849488471 @default.
• W2022026424 cites W1951218128 @default.
• W2022026424 cites W1968706475 @default.
• W2022026424 cites W1989384629 @default.
• W2022026424 cites W1989881773 @default.
• W2022026424 cites W1994556000 @default.
• W2022026424 cites W1995797967 @default.
• W2022026424 cites W2022081467 @default.
• W2022026424 cites W2046968637 @default.
• W2022026424 cites W2057611447 @default.
• W2022026424 cites W2060015752 @default.
• W2022026424 cites W2070053893 @default.
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