©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Specific Sequence of the Laminin 2 Chain Critical for the Initiation of Heterotrimer Assembly (*)

(Received for publication, October 4, 1994; and in revised form, November 9, 1994)

Atsushi Utani Motoyoshi Nomizu Satoru Sugiyama Shingo Miyamoto Peter P. Roller (1) Yoshihiko Yamada

From the Laboratory of Developmental Biology, NIDR, and Laboratory of Medicinal Chemistry, NCI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Triple-stranded laminin molecules assemble via an alpha-helical coiled-coil structure spanning approximately 600 amino acid residues of each chain. We reported that the C termini of the beta1 and 1 chains direct the specific dimer and trimer assembly (Utani, A., Nomizu, M., Timpl, R., Roller, P. P., and Yamada, Y.(1994) J. Biol. Chem. 269, 19167-19175). In this study, we focused on the mechanism of trimer formation of the alpha2 chain utilizing three different approaches. First, competition assays using mutated recombinant alpha2 chain defined a 25-amino acid sequence at the C terminus of the long arm as an essential site for assembly with beta1 and 1 chain. Site-specific mutations and synthetic peptides of this site revealed that both positively charged amino acid residues and the alpha-helical structure within this site were critical. Second, overexpression studies of recombinant alpha2 chain long arm confirmed that the C-terminal end was critical for the trimer assembly within NIH 3T3 cells. Third, circular dichroism spectroscopic examination of the complexes reconstituted in vitro revealed dynamic conformational changes of the alpha2 and 1 chains in the process of assembly. These studies also revealed that the proper folding of the extreme C terminus of alpha2 chain was critical for the stability of trimer. From these data, it is concluded that the C terminus of alpha2 chain long arm is required for the effective initiation of laminin heterotrimer assembly.


INTRODUCTION

Laminin comprises a family of glycoproteins specific to basement membrane. Laminin plays an important role in both development and maintenance of tissues. In culture, laminin has been shown to have a number of biological activities such as promoting cell adhesion, migration, growth, and neurite outgrowth (Martin et al., 1988, Beck et al., 1990). The prototypical laminin-1 molecule from mouse Engelbreth-Holm-Swarm (EHS) (^1)tumor consists of three different chains: alpha1, beta1, and 1 chains. Two other laminin chains have been identified: alpha2 chain (Am chain) in muscle, nerve, and placenta (Ehrig et al., 1990) and beta2 chain (B1s chain) originally in neuromuscular junctions (Hunter et al., 1989). The alpha2 and beta2 chains are also able to form a heterotrimer (Engvall et al., 1990; Sanes et al., 1990). More recently, the epithelial cell-specific laminin, kalinin/nicein (laminin-5, alpha3/beta3/2) and k-laminin (laminin-6, alpha3/beta1/1), has been described (Rousselle et al., 1991; Kallunki et al., 1992; Marinkovich et al., 1992).

Electron microscopy showed that laminin has a cross-like shape with one long arm and three short arms (Timpl, 1989; Engel, 1993). The long arm of laminin chain consists of about 600 amino acids of alpha-helical structure that participates in a heterotrimer formation. The alpha-helical region contains numerous heptad repeats in which hydrophobic and charged amino acids are often located at regular intervals (Paulsson et al., 1985; Beck et al., 1990, 1993). Hunter et al.(1990, 1992) reported that the proteolytic fragment (E8) of EHS laminin containing the C-terminal one-third of the long arm could be reconstituted into a heterotrimer after denaturation. Recently, we demonstrated that the specific region and the charged amino acids of the C termini of the beta1 and 1 chains were essential for the specific dimer and trimer assembly (Utani et al., 1994).

In this report, we expressed recombinant laminin alpha2 chain fragments in bacterial cells to determine the active region for trimer assembly. In addition, we expressed recombinant alpha2 chain fragments in eukaryotic cells that express endogenous beta1 and1 chains and monitored trimer assembly. Additional analysis using competitive affinity chromatography assays, in vitro reconstitution assays, and thermal stability studies were employed to analyze the active site for trimer formation. Circular dichroism spectroscopy revealed that the drastic changes of alpha-helical structure of alpha2 and 1 monomers after assembly into dimeric and trimeric structure. Furthermore, the activities of the alpha1 and alpha2 chains for trimer formation were compared. The data in this report support the model that only a limited region is necessary and sufficient to initiate the interaction with corresponding regions of the beta1 and 1 chains, resulting in the entire assembly of the laminin long arm portion.


MATERIALS AND METHODS

Expression of Recombinant Proteins

cDNAs were obtained from the published alpha1, beta1, and 1 (Sasaki and Yamada, 1987; Sasaki et al., 1987, 1988) and alpha2 chain cDNA clones (Bernier et al. 1994). The cDNA fragments were subcloned into the PQE bacterial expression vector (Qiagen, Chatsworth, CA). Recombinant proteins were expressed and purified as described previously (Utani et al., 1994). These recombinant proteins include: alpha2-400, the N-terminal 400 amino acid residues 1,575-1,974 of the alpha2 chain; alpha2-221, the C-terminal 221 amino acid residues 1,929-2,149 of the alpha2 chain; alpha2-101, 101 amino acid residues from 1,929 to 2,029 of the alpha2 chain; alpha2-120, 120 amino acid residues from 2,030 to 2,149 of the alpha2 chain; alpha1-211, the C-terminal 211 amino acid residues 1,923-2,133 of the alpha1 chain long arm; beta1-217, the C-terminal 217 amino acid residues 1,570-1,786 of the beta1 chain; and 1 -217, the C-terminal 217 amino acid residues 1,391-1,607 of the 1 chain. Additional constructs containing truncation or substitution mutations for the PQE vector were prepared using synthetic oligonucleotides and polymerase chain reaction. These constructs include; alpha2-Delta1 (residues 2,030-2,137 of the alpha2 chain), alpha2-Delta2 (residues 2,030-2,134), alpha2-Delta3 (residues 2,030-2,127), alpha2-Delta4 (residues 2,030-2,118), alpha2-S1 (residues 2,030-2,149 with a proline substitution at valine 2,144), alpha2-S2 (residues 2,030-2,149 with a proline substitution at isoleucine 2,140), alpha2-S3 (residues 2,030-2,149 with a proline substitution at isoleucine 2,126), alpha2-Delta1S1 (residues 2,030-2,137 with an alanine substitution at arginine 2,134), alpha2-Delta1S2 (residues 2,030-2,137 with an alanine substitution at lysine 2,127), alpha2-Delta1S3 (residues 2,030-2,137 with double alanine substitutions at arginine 2,134 and lysine 2,127). All constructs used in these studies were confirmed by manual DNA sequence analysis (Sequenase version 2.0, U.S. Biochemical Corp.) using [S]dATP (ICN, Costa Mesa, CA).

Peptide Synthesis

The C-terminal end of the alpha2 chain long arm was synthesized: alpha2-p55, 55 amino acid residues (2,095-2,149); alpha2-p48, 48 amino acid residues (2,102-2,149); and alpha2-p40, 40 amino acid residues (2,110-2,149). They were prepared by the solid phase peptide synthesizer (Applied Biosystems model 431A) and purified by reverse phase high performance liquid chromatography (Nomizu et al., 1992). Concentrations of the recombinant proteins and the synthetic peptides used for the in vitro reconstitution assays were determined by amino acid analysis. The concentration of the other recombinant proteins was determined by the BCA protein assay (Pierce).

Affinity Chromatography and Competition Assay

Recombinant proteins alpha2-400, alpha2-221, alpha2-101, and alpha2-120 were coupled to CNBr-activated Sepharose beads at 3 mg/10 ml (Pharmacia Biotech Inc.). Affinity chromatography assay with [S]methionine-labeled RD cell (ATCC, Bethesda, MD) lysates were performed as described by Utani et al.(1994). For competition assays increasing amounts of recombinant proteins or synthetic peptides were added into the incubation mixture of the alpha2-221-coupled beads and cell lysates. The bound materials to the alpha2-221-coupled beads were analyzed by 4% SDS-PAGE gel. After developing fluorography, the intensities of the beta1 and 1 chain bands were evaluated by densitometric scanning (LKB 2400 GelScanXL, LKB). The amounts of beta1 and 1 chains bound to affinity beads in the absence of competitors were taken as 100%. IC indicates the concentration that inhibited 50% of the binding of beta1 and 1 chains to the alpha2-221-coupled affinity beads.

Chimera Protein Expression

The cDNAs for laminin alpha2 chain long arm fragments were obtained by reverse transcriptase-polymerase chain reaction from total RNA of mouse newborn heart, using specific oligonucleotides with NaeI and SalI linkers. The wild type and a set of mutated cDNAs of the alpha2 chain long arm were subcloned into the mammalian expression vector containing an extracellular domain portion of the human interleukin 2 receptor (IL2R) as a tag. The transmembrane and intracellular portions of the receptor were deleted by the NaeI and XhoI digestion (LaFlamme et al., 1992). The constructs include: alpha2-I, the entire 575-amino acid long arm region (1,575-2,149); alpha2-II, N-terminal 400 amino acid residues (1,575-1,974); alpha2-III, the C-terminal 221 amino acid residues (1,929-2,149); alpha2-IV, the 12-amino acid residue deletion (residues 1,575-2137); alpha2-V, 575 residues (1,575-2,149) with a proline substitution at isoleucine 2,126; and alpha2-VI, 575 residues with a proline substitution at isoleucine 2,140. The expression was regulated under the control of the cytomegalovirus promoter. Two µg of each of these constructs were transfected into NIH 3T3 cells (2.0 times 10^5) (ATCC, Bethesda, MD) in a six-well dish using 6 µl of LipofectAMINE for 6 h (Life Technologies, Inc.). Twenty-four h after transfection, the cells were labeled with [S]methionine at 50 µCi/ml in methionine-free Dulbecco's modified Eagle's medium for 4 h. Cell lysates were prepared using RIPA (0.1% SDS, 1% Nonidet P-40, 0.5% deoxycholate, Tris-HCl, pH 8.0) buffer instead of Triton X-100/PBS buffer. Immunoprecipitation was performed with anti-IL2R antibody (0.2 µg/ml) (T cell Diagnostics, Cambridge, MA) and protein G beads. Western blotting analysis was done using the mixture of polyclonal anti-alpha2-400 and anti-alpha2-221 antibodies and chemiluminescence (ECL, Amersham Corp.).

In Vitro Reconstitution Assay

Reconstitution of laminin trimer complexes in vitro was done as described by Utani et al.(1994).

Circular Dichroism (CD) Analysis and Thermal Stability

CD analysis of the reconstituted complexes was recorded in PBS, pH 7.4, at 1.5 µM protein concentration in the range of 195-250 nm. A Jasco model J-500A/DP-501N CD spectropolarimeter equipped with Hellma cell of 1 mm path length was used (Nomizu et al., 1992). The contents of alpha-helical structure were calculated by the formula of Chen and Yang (Chen et al., 1974). Thermal transition curves of the reconstituted products were obtained by monitoring ellipticity at 222 nm in the temperature range 25-80 °C in both directions. The degree of conversion (F) was calculated from F = ([] - [](d))/([](n) - [](d)) where [] is the ellipticity at the indicated temperature. [](n) and [](d) are the ellipticities of the native and fully unfolded proteins, respectively. T(m) was determined as a temperature indicating F = 0.5.


RESULTS

Affinity Chromatography Assay Revealed the Region of alpha2 Chain Involved in the Trimer Assembly

Previously, we showed that bacterial recombinant proteins from the C-terminal 221 amino acids of the long arm of the alpha2 chain was able to form a trimer with the native beta1 and 1 chains using an affinity chromatography assay (Utani et al., 1994). In the present work, we have generated a set of cDNA constructs covering the entire alpha2 chain long arm and coupled the expressed recombinant proteins to affinity beads (Fig. 1, left). The affinity beads were tested for their activity by incubation with [S]methionine-labeled RD cell lysates and followed by SDS-PAGE and fluorography. Two recombinant proteins immobilized on beads, alpha2-221 and alpha2-120, were active for assembly with the endogenous beta1 and 1 chains (Fig. 1, right, lanes2 and 5). These results indicate that one site active for trimer assembly exists in the C-terminal 120-amino acid region of the alpha2 chain long arm.


Figure 1: Trimer formation of recombinant alpha2 chain with beta1 and 1 chains in the affinity bead assays. Left, the schematic picture of laminin-2 and the bacterially expressed recombinant proteins of truncated alpha2 chain long arm. The long arm of alpha2 chain spans 575 amino acid residues between the last cysteine residue of the short arm region and the first cysteine residue of the globular region. The recombinant proteins contain the following region of the alpha2 chain: alpha2-400, residues 1,575-1,974; alpha2-221, residues 1,929-2,149; alpha2-101, residues 1,929-2,029; alpha2-120, residues 2,030-2,149. Right, the recombinant proteins immobilized on CNBr-activated Sepharose beads were incubated with [S]methionine-labeled RD cell lysates. The bound proteins were analyzed by 4% SDS-PAGE under reducing conditions followed by fluorography. Lane 1, immunoprecipitates of RD cell lysates with anti-EHS laminin antibody showing beta1 and 1 chains; lane 2, bound materials to the alpha2-221 affinity beads; lane 3, the alpha2-400 affinity beads; lane 4, the alpha2-101 affinity beads; lane 5, the alpha2-120 affinity beads. Molecular markers are shown on the left in kDa.



Delineation of the Active Site for Trimer Assembly by Competition Assay

We wished to map more precisely the minimal region within the alpha2 chain responsible for assembly. The truncated or substituted mutant recombinant alpha2 chains or synthetic peptides were tested for inhibitory activity as soluble competitors on trimer formation using the immobilized alpha2-221 affinity beads (Fig. 2A). The activity in the trimer assembly was estimated by measuring the quantity of the beta1 and 1 chains following fluorography. Some of the competitors showed dose-dependent inhibitory activity (Fig. 2B). Since the alpha2-120 recombinant protein showed the same ability to block heterotrimer formation as the alpha2-221 (IC = 2.0 nM), the active site was thought to exist in the alpha2-120 protein as indicated by the affinity chromatography assays. Additional deletion mutants of alpha2-120, including alpha2-Delta1, alpha2-Delta2, alpha2-Delta3, and alpha2-Delta4 showed progressively less trimer assembly activity as more amino acid residues were deleted from the C terminus (Fig. 2B). For example, alpha2-Delta1 and alpha2-Delta2 had IC values of 16 and 580 nM, respectively. alpha2-Delta3, a 22-amino acid C terminus deletion, still retained activity but was 3,000-fold weaker than that of the alpha2-120. Finally, further deletion as in alpha2-Delta4, showed a complete inability to interact with the beta1 and 1 chains at concentrations up to 10 µM. These results suggest that the C-terminal 31-amino acid sequence between the residues 2,119 and 2,149 of the alpha2 chain is prerequisite for forming the heterotrimer. The activity of alpha2-Delta1 was only 8-fold weaker than alpha2-120, suggesting the most C-terminal 12-amino acid region (2,138-2,149) may not be critical compared with the N-terminal sequence adjacent to this site.


Figure 2: Inhibitory activity of various mutants and synthetic peptides of alpha2 chain on trimer formation with beta1 and 1 chains. A, a list of the deletion and substitution mutants, and synthetic peptides of alpha2 chain long arm and the values of IC. The C-terminal amino acid sequence is shown at the top. IC (nanomolar) indicates the concentration of the competitor that inhibited 50% of the binding of the beta1 and 1 chains to the alpha2-221-coupled beads. n.d., not determined. The values of IC were > 6,000 nM for alpha2-S3 and >3,300 nM for alpha2-p48 and alpha2-p40. B, inhibitory activities of deletion mutants as soluble competitors. x axis shows the concentration of the competitors. y axis indicates the percent (%) binding of beta1 and 1 chains to the alpha2-221-coupled affinity beads. The amounts of those bound to affinity beads in the absence of competitors were taken as 100%. Each point shows the mean value obtained by densitometric scanning of the x-ray films (as seen in Fig. 7A) of the two or three independent experiments. The competitors are listed at right.




Figure 7: Comparison of the activity for trimer formation of alpha2 and alpha1 chain. A, binding of beta1 and 1 chains to the alpha2-221-coupled affinity beads were competed with the increasing amounts of the alpha2-221 (lanes 1-5) or those of the alpha1-211 (lanes6-10). The concentrations of the competitors are:(-) in lanes 1 and 6, 25 nM in lanes2 and 7, 50 nM in lanes3 and 8, 250 nM in lanes4 and 9, and 1.25 µM in lanes5 and 10. The materials bound to the alpha2-221-coupled affinity beads were analyzed by SDS-PAGE and subsequent fluorography. B, amino acid sequence homology among the alpha2, alpha1, and alpha3 chains. The numbers of amino acid residues of mouse alpha2, alpha1, and human alpha3 chain are from Bernier et al.(1995), Sasaki et al.(1989), and Ryan et al.(1994), respectively. Dottedboxes show identical sequences, and d indicates the position of the heptad repeats of the hydrophobic residues within the C termini. Arrow depicts the 25-amino acid region, and asterisks indicate 3 amino acids critical for trimer formation of alpha2 chain defined by competition assay.



To verify this observation, a proline substitution within this 12-amino acid region was introduced at the hydrophobic residue to disrupt the alpha-helical structure. alpha2-S1 did not significantly reduce the activity in this competition assay, since the IC value was 2.4 nM (Fig. 2A). A different proline substitution, alpha2-S2, reduced the activity by approximately 3-fold with an IC value of 6.2 nM. These data are consistent with those presented above, in which truncated mutants from this region showed little loss of trimer assembly activity. Finally, a proline substitution at isoleucine 2,126 (alpha2-S3) was completely inactive as a competitor (Fig. 2A). These data strongly suggest that the conformation surrounding isoleucine 2,126 is critical for trimer assembly, consistent with the precipitous drop in activity seen with alpha2-Delta3. Based on the results from the alpha2-Delta4 and alpha2-S1, it was concluded that the C-terminal 25-amino acid sequence (residues 2,119-2,143) was the essential site for trimer assembly.

We prepared the synthetic peptides consisting of C terminus of the alpha2-120 to further delineate the active region (Fig. 2A). The activity for trimer assembly of the synthetic peptides in the competition assay was surprisingly weak compared with that of alpha2-120 (Fig. 2A). For example, peptide alpha2-p55, which is the strongest competitor of the synthetic peptides, was approximately 1,700-fold weaker than alpha2-120. The shorter peptide alpha2-p40 was completely inactive, despite containing the critical 25-amino acid region determined by the competition assays using recombinant proteins. As discussed later, the low contents of alpha-helical structure of the short synthetic peptides may explain the poor activity as competitor. Thus, synthetic peptides were not helpful in identifying the active site by the competition assay.

The Positively Charged Amino Acids of the Critical Region

A helical wheel alignment analysis of the C-terminal end of alpha2 chain long arm revealed that there were 3 conserved positively charged amino acid residues located at the ``e'' position of alpha2 and alpha1 chains (Fig. 7B). Since lysine 2,141 is located on the C-terminal side of the replaced isoleucine 2,140 in alpha2-S2, which reduced activity only 3-fold, we selected 2 other positively charged residues for the alanine substitution (Fig. 3). Three new recombinant proteins were generated using alpha2-Delta1 as a starting construct in order to examine the role of these 2 charged amino acid residues in trimer formation. The alanine substitution mutants included arginine 2,134 (alpha2-Delta1S1), lysine 2,127 (alpha2-Delta1S2), and a double mutation at both sites (alpha2-Delta1S3) (Fig. 3). alpha2-Delta1S1 had an IC value of 50 nM compared with the IC value of 16 nM for alpha2-Delta1. However, alpha2-Delta1S2 had a marked loss in trimer assembly activity (30-fold) with an IC value of 550 nM. The IC value of the double alanine substitutions at both positions (alpha2-Delta1S3) was 5,000 nM. This double substitution was approximately 300-fold less active than the alpha2-Delta1, suggesting that both these charged amino acids are important for the activity for assembly with beta1 and 1 chains.


Figure 3: Inhibitory activities of substitution mutants of the two positively charged amino acids. A list of the alanine substitution mutants of the positively charged amino acids derived from the alpha2-Delta1. d indicates the hydrophobic residue in the heptad repeat (abcdefg). The values of IC (nanomolar) are shown at right.



Transfection Study of the alpha2 Chain with NIH 3T3 Cells

We tried to confirm whether the essential site identified by the competition assay was active under physiological conditions. We utilized NIH 3T3 cells that showed a high expression of the proteins following transfection of cDNA under the control of cytomegalovirus promoter. NIH 3T3 cells produce endogenous beta1 and 1 chains, confirmed by the immunoprecipitation with polyclonal anti-laminin-1 antibody (data not shown). The intact long arm and truncated or substituted mutants of alpha2 chain were designed to fuse to the extracellular domain of IL2R cDNA (Fig. 4A). The overexpressed recombinant alpha2 chains were immunoprecipitated with anti-IL2R antibody and analyzed by 5% SDS-PAGE under reducing conditions (Fig. 4B). The transfected proteins were verified by Western blotting with an anti-alpha2 chain antibody (Fig. 4B, middlepanel). alpha2-I and alpha2-III proteins showed activity for the assembly as proved by the presence of co-precipitated beta1 and 1 chains (Fig. 4B, lanes7 and 8). The N-terminal 400-amino acid portion of the long arm (alpha2-II) showed no activity, although a similar level of transfected protein was expressed (Fig. 4B, lane3 of upper and lowerpanels). These results confirm that a region of 221 amino acids of the C terminus of the alpha2 chain long arm can independently form a stable heterotrimer with the beta1 and 1 chains under physiological conditions.


Figure 4: Trimer formation with transfected alpha2 chains in NIH 3T3 cells. A, the entire and mutated long arm of the alpha2 chains were constructed using the cytomegalovirus promoter driven mammalian expression vector. Each construct has an extracellular domain of the 55-kDa IL2R at N terminus as a tag (shown as a dottedcircle). Asterisks show the substitution sites within alpha2-V and alpha2-VI. B, 24 h after transfection with LipofectAMINE, cells in a six-well dish were labeled with [S]methionine. Expressed proteins were immunoprecipitated from cell lysates with monoclonal anti-IL2R antibody and protein G beads and analyzed by 5% SDS-PAGE under reducing conditions. Immunoprecipitates from the cell lysate transfected with alpha2-I (lanes 1 and 7), untransfected cells (lane 2), alpha2-II (lane 3), alpha2-IV (lane 4), alpha2-V (lane 5), alpha2-VI (lane 6), and alpha2-III (lane 8). Upper panel, long exposure of the fluorography to show beta1 and 1 chains. Lower panel, short exposure of the same x-ray film of upper panel. Middle panel, Western blotting probed with anti-alpha2 antibodies. Arrow indicates doublets of beta1 and 1 chains. Asterisks show nonspecifically precipitated fibronectin. Molecular markers are shown at left.



To verify the active site identified by the competition assays, three more mutant cDNAs were prepared. alpha2-IV included nearly the whole alpha-helical region but with the same C-terminal deletion as the alpha2-Delta1. alpha2-V and alpha2-VI had the entire alpha-helical region but with the same proline substitution as the alpha2-S3 and alpha2-S2, respectively (Fig. 4A). Among these three proteins, only alpha2-VI showed a weak activity for trimer assembly with the beta1 and 1 chains (Fig. 4B, lane6, upperpanel). These results are consistent with the observations that the alpha2-S2 and alpha2-Delta1 reduced the activity in the competition assays by approximately 3- and 8-fold, respectively (Fig. 2A). Thus, the C terminus of the alpha2 chain long arm, including the residues surrounding isoleucine 2,126, is critical for trimer assembly in the transfected cells.

In Vitro Reconstitution Assays and Thermal Stability of the Reconstituted Trimer Complexes

In vitro reconstitution assays were utilized to identify the active site for trimer formation. A set of mutated recombinant alpha2 chains or synthetic peptides were reconstituted with equimolar amounts of recombinant protein beta1-217 and 1-217. The C-terminal 217-amino acid residues of beta1 and 1 chain were shown to form a heterotrimer with alpha2-211 and alpha2-120 in the previous study (Utani et al., 1994). The reconstituted products were analyzed by 4% non-denaturing gel electrophoresis (Fig. 5). Reconstituted trimeric complexes (Fig. 5, upper arrow) migrated slower than the dimeric complex of beta1-217/1-217 (lowerarrow). The assembly activity was completely lost in the 31- and 22-amino acid deletion from the C terminus of the alpha2-120 (alpha2-Delta4 and alpha2-Delta3; Fig. 5, lanes4 and 5) and in the proline substitution at isoleucine 2,126 (alpha2-S3, Fig. 5, lane9). In addition, alpha2-101 was inactive consistent with the data of affinity chromatography (Fig. 5, lane3; Fig. 1, lane 4). The rest of recombinant alpha2 chain proteins were active. The synthetic peptide alpha2-p55 was active but alpha2-48 was only weakly active for the trimer formation as there remained a detectable amount of the beta1-217/1-217 dimer (Fig. 5, lanes 16 and 17). alpha2-p40 was completely inactive (Fig. 5, lane 15). Compared with the activities observed in the competition assays, the IC value of 5,000 nM of the alpha2-Delta1S3 was the lowest value for the complete achievement of trimer formation under the conditions employed here.


Figure 5: In vitro reconstitution assays of trimer assembly. A set of recombinant alpha2 chains or synthetic peptides were mixed with equimolar amounts of beta1-217 and 1-217. The products were analyzed by 4% non-denaturing electrophoresis and stained with Coomassie Brilliant Blue. The alpha2 components of the mixture are listed on the top of each lane. The monomer of the alpha2 chain components could not enter the gel except alpha2-101 (lane 3) (Utani et al., 1994). Upperarrow indicates the trimer complexes, and lowerarrow indicates beta1-217/ 1-217 heterodimer.



The thermal stability of the trimeric complexes was evaluated by monitoring the [] by CD spectroscopy during the heat change from 25 °C to 80 °C (Table 1). The three different kinds of trimers with the alpha2-221, alpha2-120, and even with the alpha2-p55 showed high T(m) values of 63-64 °C. This is in good agreement with the T(m) value obtained with the proteolytic fragment E8 of laminin-1 (Paulsson et al., 1985; Hunter et al., 1992), suggesting these trimer formed a coiled-coil trimeric structure. These results indicated that the C-terminal 55 amino acid residues of the alpha2 chain long arm was able to form a stable trimer with beta1-217/1-217. On the contrary, the T(m) values of the reconstituted trimers with alpha2-Delta1, alpha2-S1, and alpha2-S2 decreased more than 10 °C. These significantly lower T(m) values suggest that the 12-amino acid sequence deleted in alpha2-Delta1 greatly contributes to the formation of the stable trimer utilizing its alpha-helical structure. As for the three alanine substitutions of the charged amino acid residues of alpha2-Delta1, these mutants showed T(m) values only 3 °C and 4 °C lower than that of the trimer with alpha2-Delta1 (51 °C). The slightly reduced T(m) value may indicate that these 2 positively charged residues contribute to some degree of stability. Furthermore, these data indicate that the drastic changes of the activities induced by the truncation or substitution mutants in the competition assay did not directly correlate with the thermal stability once the trimers were formed.



Conformational Changes after Assembly into Dimer and Trimer

Structural analysis of reconstituted complexes was performed using CD spectroscopy at 25 °C (Fig. 6). Lower ellipticity at 222 nm ([]) indicates higher alpha-helix content (Chen et al., 1974). The CD spectrum of beta1-217 monomer showed a strikingly high alpha-helix content of 75%. However, 1-217 and alpha2-221 exhibited much lower alpha-helix content, 29% and 34%, respectively (Fig. 6A). Furthermore, the [] of beta1-217/1-217 dimer was significantly lower than the mean [] value of beta1-217 and 1-217 monomers (beta1-217 + 1-217) (Fig. 6B). An alpha-helix content of the dimer (beta1-217/1-217) and the mean content of monomers (beta1-217 + 1-217) were 75% and 52%, respectively. This high negative ellipticity (-26.9 times 10^3(degreebulletcm^2bulletdmol)) of beta1-217/1-217 dimer was consistent with that of beta1 and 1 dimer derived from the proteolytic E8 fragment (-28 times 10^3(degreebulletcm^2bulletdmol)) (Hunter et al., 1992). These data indicated that the complex of recombinant proteins beta1-217 and 1-217 formed a coiled-coil heterodimer. The increase in alpha-helix content from 52% to 75% after dimer formation suggests that 1-217 significantly changed its conformation since it shows only 29% alpha-helix content as a monomer. A trimer, beta1-217/1-217/alpha2-221, showed notably higher alpha-helix content (74%) than the mean alpha-helix content (46%) of the monomers (beta1-217 + 1-217 + alpha2-221) (Fig. 6C). These results suggest that 1-217 and alpha2-221 drastically increases the alpha-helix content after trimer formation. Our previous report suggests that beta1/1 dimer is formed before trimer assembly since alpha2 chain cannot assemble with beta1 or 1 monomers (Utani et al., 1994). If this is the case, the difference between the [] of (beta1-217/1-217/alpha2-221) and that of (beta1-217/1-217 + alpha2-221) represents the changes of the beta1-217/1-217 dimer and/or alpha2-221 monomer (Fig. 6C). Since alpha2-221 has only 34% alpha-helix content as a monomer in comparison to beta1-217/1-217 dimer (75%), it is likely that alpha2-221 drastically increases its alpha-helix content after trimer formation. Thus, the two components, 1-217 and alpha2-221, increased their alpha-helix content after assembly into a coiled-coil structure, although beta1-217 exhibited high alpha-helix content as a monomer.


Figure 6: Circular dichroism analysis of alpha-helical structure. CD spectra of monomer and reconstituted complexes were monitored in PBS at protein concentrations of 1.5 µM. A, spectra of monomer, beta1-217, 1-217, and alpha2-221. B, spectra of dimer (beta1-217/1-217) and mean of monomer s(beta1-217 + 1-217). C, spectra of trimer (beta1-217/1-217/alpha2-221), mean of the dimer and monomer (beta1-217/1-217 + alpha2-221), and mean of the monomers (beta1-217 + 1-217 + alpha2-221).



Comparison of the Trimer Formation Activity between the alpha1 and alpha2 Chains

Since both the alpha1 and alpha2 chains assemble with the beta1 and 1 chains (Engvall et al., 1990; Utani et al. 1994), it is of interest to know whether there is a difference in assembly activity. Competition assays were performed using alpha2-221 and alpha1-211 as soluble competitors to alpha2-221 immobilized affinity beads (Fig. 7A). There was no significant difference between both competitors. Both competitors showed the same IC values of 2.0 nM. An alignment of amino acid sequence of both the alpha1 and alpha2 chains reveals that there is strong sequence conservation in the C-terminal 50 amino acids, while the overall sequence identity of the entire long arm region is much lower (Bernier et al., 1995) (Fig. 7B). These findings suggest that alpha1 chain also utilizes the conserved C terminus of the long arm as an active site for trimer assembly with beta1 and 1 chains.


DISCUSSION

Laminin chains have a large alpha-helical region of about 600 amino acids in their long arm where the three chains interact and form a triple-stranded coiled-coil structure (Paulsson et al., 1985). One of the major questions that we wished to address in the present studies was what region of the alpha2 chain long arm was required for laminin assembly. Furthermore, we discussed mechanisms for the initiation of laminin heterotrimer formation.

Here, we employed three different approaches to evaluate the essential region of the alpha2 chain for trimer assembly: competition assays, transfection assays, and in vitro reconstitution assays. Each of these assays has unique properties and different sensitivity. The competition assay for trimer assembly proved most useful to map precisely the active region of the alpha2 chain. Deletion and substitution mutants allowed us to define a sequence within the C terminus of the alpha2 chain long arm that is essential for trimer assembly. The data indicating progressive loss of activity as more amino acid residues were deleted from the C terminus suggest that all of these amino acids, but especially a 25-amino acid sequence (residues 2,119-2,143), act in unison to promote trimer assembly. The transfection assay showed that the alpha2-IV construct with a 12-amino acid deletion at the C terminus had little assembly activity. However, the alpha2-Delta1 construct with the same deletion at the C terminus retained some activity in the competition assays. Therefore, it seems impracticable to determine the precise activity of the mutated alpha2 chain with less activity for the assembly in the transfection assays. However, the observation that the 12-amino acid deletion and the disruption of the alpha-helical structure within the C terminus abrogated the activity for trimer formation reinforces the significance of the C-terminal 25-amino acid sequence as an essential site even under physiological conditions.

In the in vitro reconstitution assay, the absence of activity using alpha2-Delta3 (a 22-amino acid deletion from C terminus) and alpha2-S3 (a proline substitution at isoleucine 2,126) demonstrated again the importance of the C-terminal region of the alpha2 chain long arm. However, differences in assembly activity observed in the competition assays could not be detected. For example, alpha2-Delta1S3 with a very low activity in the competition assays showed activity for trimer formation in the in vitro reconstitution assays. These results may be due to the high concentrations of the substrates used in the in vitro reconstitution assays, which make it difficult to distinguish the relevant activity of the mutant alpha2 chains and synthetic peptides with less activity. Nonetheless, the reconstituted trimer complexes gave us substantial information about the conformation and thermal stability requirements of laminin chain assembly as discussed below.

Charged amino acids at ``e'' and ``g'' positions of the heptad repeat have been shown to play an important role in heterodimer of the b-Zip family of transcription factors (Schuermann et al., 1991; O'Shea et al., 1992). By calculating the electrostatic interactions between the charged amino acids of each laminin chain, Beck et al. (1990, 1993) have suggested that ionic interactions may play a crucial role in chain-specific laminin assembly. Alanine substitutions for the positively charged amino acids within the critical site of the alpha2 chain markedly reduced its trimer formation activity. These results, together with our earlier observations that alanine substitution of the glutamate residue at the C terminus of the beta1 chain abolishes trimer formation (Utani et al., 1994), provide evidence that electrostatic interactions play a critical role in triple-stranded coiled-coil formation of laminin.

It is noteworthy that alpha2-p40, a short peptide, was inactive in the competition assays and in the in vitro reconstitution assays, despite that it encompasses the essential 25-amino acid sequence for trimer assembly. Furthermore, alpha2-p55 is a poor competitor (IC = 3,300 nM) when compared with alpha2-120 (IC = 2.0 nM) in the competition assays. CD analysis revealed that the synthetic peptides with length shorter than 40 amino acids from the C terminus of the alpha2 chain long arm did not retain alpha-helical structure but had a random coil structure (data not shown). The observation that the C terminus of alpha2 chain long arm requires long stretches of amino acid residues for the alpha-helical structure could explain the low activity of the short peptides in the competition assays.

The thermal stability studies provided additional insights into the mechanism of trimer assembly. Although alpha2-p55 was a poor competitor in the competition assay, alpha2-p55 was able to form a trimer as well as alpha2-120 or alpha2-221. It is likely that once the initial interaction occurs with beta1 and 1 heterodimer, alpha2-p55 undergoes a conformational change on assembly into the stable trimer structure, which is indistinguishable from that of the trimer seen with alpha2-120 or alpha2-221. The drastic increase of the alpha-helix content of alpha2-221 after trimer formation may support this hypothesis. Based on the data from CD analysis, both alpha1-211 and the C-terminal 223-amino acid residues of 2 chain (Utani et al., 1994) showed a low alpha-helix content of 27% and 29%, respectively. Since the 1 and alpha2 chains also have a low alpha-helix content as monomers, the drastic increases of an alpha-helix content of the alpha and chains after assembly may be a common feature to laminin dimer and trimer formation. The stability of the trimer with the alpha2-S1 (a proline substitution at valine 2,144) significantly decreased despite that it retained almost complete competition activity. These observations may be also explained by the conformational change of alpha2 chain after trimer formation. The alpha-helical structure at the extreme C terminus does not affect the initiation of trimer formation, which in turn becomes essential for maintaining the stability with an increased alpha-helix structure after trimer formation. A similar mechanism may also explain the equivalent T(m) values of the trimers with three different alanine substitution mutants. Thus, electrostatic interactions are an essential part of the assembly process but lose their significance after drastic conformational change as a result of trimer formation.

A recently identified alpha3 chain also forms a trimer with beta1 and 1 chain as a laminin-6 molecule (Marinkovich et al., 1992). Alignment of the C-terminal sequence of human alpha3 chain long arm (Ryan et al., 1994) reveals striking sequence identity within the essential 25-amino acid site for assembly (Fig. 7B). Furthermore, three essential residues for the activity are conserved, i.e. isoleucine at 2,126, lysine at 2,127, and arginine at 2,134. These findings suggest that alpha3 chain may also use this region for trimer formation. Recently, Harbury et al.(1993, 1994) reported that synthetic peptides with isoleucine residues at ``a'' and ``d'' positions of leucine zipper motif of b-Zip protein could form a coiled-coil homotrimer. When leucine substitutions were introduced at all isoleucine residues of alpha2-p55, this mutant reduced its trimer formation activity (Nomizu et al., 1994). Therefore, the two conserved isoleucine residues at ``a'' and ``d'' positions (2,126 and 2,130) within the essential site may contribute to form a trimer with the isoleucine residues concentrated in the C terminus of 1 chain. In this context analysis of the active site of alpha1 and alpha3 chains will deepen our understanding of the mechanisms by which laminin form a triple-stranded coiled-coil structure.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: EHS, Engelbreth-Holm-Swarm; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; IL2R, interleukin 2 receptor.


ACKNOWLEDGEMENTS

We thank Peter Burbelo for helpful comments. We also thank Maura Kibbey and Jeffrey B. Kopp for critical reading of the manuscript.


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