©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Laminin Interactions Important for Basement Membrane Assembly Are Promoted by Zinc and Implicate Laminin Zinc Finger-like Sequences (*)

(Received for publication, October 10, 1995)

John B. Ancsin Robert Kisilevsky (§)

From the Department of Pathology, Queen's University, Kingston, Ontario, K7L 3N6 Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Laminin is an abundant basement membrane (BM) glycoprotein which regulates specific cellular functions and participates in the assembly and maintenance of the BM superstructure. The assembly of BM is believed to involve the independent polymerization of collagen type IV and laminin, as well as high affinity interactions between laminin, entactin/nidogen, perlecan, and collagen type IV. We report here that Zn can influence laminin binding activity, in vitro. Laminin contains 42 cysteine-rich repeats of which 12 contained nested zinc finger consensus sequences. Recently, the entactin binding site was mapped to one of these zinc finger-containing repeats on the laminin chain (Mayer, U., Nischt, R., Poschl, E., Mann, K., Fukuda, K., Gerl, M., Yamada, Y., and Timpl, R.(1993) EMBO J. 12, 1879-1885). Based on these observations, the effect of a series of essential ions (Ca, Cd, Cu, Mg, Mn, and Zn) on laminin binding activity was evaluated. Zn was found to be the most effective at enhancing laminin-entactin and laminin-collagen type IV binding. Laminin-bound Zn was detected by flame atomic absorption spectroscopy at a maximum of 8 mol/mol of laminin. Furthermore, Ca-dependent laminin polymerization was unaffected by Zn, an observation consistent with the lack of zinc finger-containing repeats in the terminal globular domains required for polymerization. We conclude that Zn-laminin complexes may generate high affinity binding sites which contribute to BM cross-linking important for its assembly and homeostasis. Zinc is likely a cofactor for 2 kinds of cross-linking interactions; one involving direct binding between laminin and collagen type IV and the other a ternary complex of laminin-entactin-collagen type IV.


INTRODUCTION

Basement membrane (BM) (^1)is a distinct type of extracellular matrix, which divides tissue into compartments, provides filtration and structural support, sequesters growth factors, and directly influences cellular behavior(2, 3) . These functions are believed to be dependent on BM composition and ultrastructure. The assembly of BM involves the synthesis and secretion of the major BM components (laminin, entactin/nidogen, perlecan, and collagen type IV) into a diffusion-limited space where, by a mass action-driven process, they become interconnected through site-specific interactions generating a 50-200 nm thick network. Little is known of the physical nature of these binding sites or of the regulatory factors which govern their interactions. Deviation in BM metabolism is believed to underlie complications associated with diseases such as Alport's and Goodpasture's syndromes(4, 5) , diabetes mellitus(6) , amyloid(7) , and Alzheimer's disease(8, 9) .

Laminin is a unique and essential component of BM, contributing to its architecture, and providing signals for cell adhesion, migration, and differentiation. The prototype of this family of glycoproteins, laminin-1, is derived from Engelbreth-Holm-Swarm (EHS) tumor and is composed of three different subunits alpha1, beta1, and 1 (previously A, B1, and B2, respectively) which form a multidomain cruciform structure possessing one long and three short arms. Several studies have shown that laminin can exist as a polymer both in vivo and in vitro. The assembly of BM involves primarily the polymerization of 2 independent networks: one of collagen type IV, which becomes covalently stabilized(10, 11, 12, 13) , and the other of laminin, in a noncovalent, calcium-dependent process(14, 15, 16) . In the BM of EHS tumor, about 80% of the laminin is deposited as an independent polymer, while the remainder is found also anchored, noncovalently, to the collagen type IV network(17) . High affinity interactions between laminin-entactin and entactin-collagen type IV have been reported supporting the concept that the 2 networks are interconnected by entactin(18, 19) . However, direct association between laminin short arms and collagen type IV has also been reported (20, 21) .

A recent study focusing on Alzheimer's disease presented data showing Zn stimulated laminin binding to the Alzheimer's amyloid precursor protein(22) . In the same report, it was observed that of the many cysteine-rich domains in laminin (also known as EGF-like repeats), some contained the zinc finger consensus sequence(23) . Of 42 cysteine-rich repeats found on the amino-terminal ends of the alpha1, beta1, and 1 chains, 12 appear to have Cys spacing similar to that observed for certain zinc fingers. More recently, the entactin binding site was localized to a 58-mer corresponding to the 4th repeat of domain III, on the 1 chain (1) which, as our observation suggests, contains a nested zinc finger sequence.

On the basis of these observations, we investigated the influence of different essential ions on 3 laminin interactions important for BM assembly. We report here that, of all the essential ions tested (Ca, Cd, Cu, Mg, Mn, and Zn), zinc was the most effective at enhancing laminin-entactin and laminin-collagen type IV interactions. The zinc effect was saturable, and a maximum of 8 mol of zinc/mol of laminin was detected by flame atomic absorption spectroscopy. Laminin polymerization was not zinc-dependent, consistent with the lack of zinc finger-like sequences in the terminal domains which are required for polymerization. Our results provide biochemical evidence supporting earlier reports (20, 21) that laminin-collagen type IV interactions could occur without entactin acting as a bridging molecule. We also provide evidence that the entactin and collagen type IV binding sites on laminin may involve a zinc finger-like secondary structure. To our knowledge, this is the first report of an extracellular zinc finger motif acting directly as, or contributing to, high affinity protein-protein interactions. It also implicates Zn as an important cofactor/modulator of BM assembly.


MATERIALS AND METHODS

Protein Purification

Laminin and entactin were purified from Engelbreth-Holm-Swarm (EHS) mouse sarcoma propagated in nonlathyritic mice (Swiss Webster, Charles Rivers, Montreal, Quebec), harvested at 2-4 cm, frozen in N(2)(l), and stored at -70 °C. Purification was based on a NaCl extraction procedure in (24) and (25) , and from Hynda Kleinman, NIH). (^2)Tumor was homogenized and washed 2 times in 3.4 M NaCl, 50 mM Tris, 2 mM EDTA, 1 mMN-ethylmaleimide, 1 mM phenylmethylsulfonyl fluoride, pH 7.5, centrifuged at 10,000 times g for 15 min, discarding the supernatant each time. The residue was extracted in the same buffer but with 0.5 M NaCl, for 4-8 h, then centrifuged as above. The supernatant was precipitated with 30% saturated ammonium sulfate, and the pellet was redissolved and dialyzed against 50 mM Tris, 150 mM NaCl, pH 7.5 (TBS). NaCl was increased to 1.7 M, precipitating collagen type IV which was removed by centrifugation. The supernatant was applied to a Bio-Gel A-5m (2.5 times 120 cm) gel filtration column eluted with TBS. Fractions containing the laminin-entactin complex were pooled, concentrated with Aquacide II (Calbiochem), and dialyzed against TBS/2 M guanidine HCl. The dialysate was applied to a Sephacryl-S400HR (2.5 times 140 cm) column eluted with the same buffer. Laminin and entactin fractions were pooled, dialyzed against TBS, concentrated, frozen in N(2)(l), and stored at -70 °C. The steps in the procedures were monitored by SDS-polyacrylamide gel electrophoresis on a 5-10% acrylamide gradient gel(26) .

Solid Phase Binding Assay

An enzyme-linked immunosorbent assay technique was employed to study the interaction between laminin, entactin, and collagen type IV. Polystyrene microtiter plates (Immulon 4, Dynatech Laboratories) were coated with 100 µl of 200 ng/ml entactin or 500 ng/ml collagen type IV in 20 mM NaHCO(3), pH 9.6. After an overnight incubation at 4 °C, the plates were washed with 20 mM Tris-HCl, 150 mM NaCl, pH 7.5 (TBS), then incubated with 1% bovine serum albumin in TBS (150 µl) for 2 h at 37 °C to block residual hydrophobic surfaces. Plates were washed with TBS containing 0.05% (w/v) Tween 20 (TBS-Tween), then laminin at different concentrations was added in the same buffer and left overnight at 4 °C to allow maximum binding. Plates were washed again in TBS-Tween and incubated with mouse anti-laminin IgG (Sigma) diluted 1:750 in TBS-Tween, 0.1% bovine serum albumin for 2 h at 37 °C, rewashed, then incubated in the same way with goat anti-rabbit IgG conjugated with alkaline phosphatase (Boehringer Mannheim) at a 1:500 dilution. After washing, bound IgG was detected by the addition of alkaline substrate solution containing 2 mg/ml p-nitrophenyl phosphate, 0.1 mM ZnCl(2), 1 mM MgCl(2), and 100 mM glycine, pH 10.0. Plates were left at room temperature for 15-30 min, and the reaction was stopped with 50 µl of 2 M NaOH. The absorbance due to the released p-nitrophenol was measured at 405 nm with a Titertek Multiscan/MCC 340 (Flow Laboratories). The amount of bound ligand was determined by subtracting the optical density of the blank wells, in which the ligand was omitted. For direct quantitation, ligand standards were precoated onto wells on the same plates as the test proteins in order to generate standard curves.

To measure coating efficiency, laminin, entactin, and collagen type IV were radioiodinated with I, using IODOBEADS (Pierce), and the amount of protein coated onto the microtiter plates was measured by subtracting the counts/min remaining in the coating buffer after coating from the total. Coating efficiency was 90-100% at the concentrations used in the binding assays.

Binding data were analyzed as in (22) with a nonlinear curve fit program (SigmaPlot, Jandel Scientific) using for a one-binding site model with nonspecific binding or for a two-binding site model with nonspecific binding, where S is the proportionality constant for nonspecific binding and L is the laminin concentration.

In all cases, the data fit the one-site model the best, and nonspecific binding was very low (S < 10).

Zinc Analysis

Laminin zinc content was assayed by flame atomic absorption spectroscopy using elemental zinc standards (0-2 ppm). Laminin was either assayed directly after purification or after loading with ZnCl(2), which involved dialysis against 1 liter of TBS containing 50 µM ZnCl(2) overnight, followed by extensive dialysis against TBS, 0.1 mM EDTA, then just TBS to remove unbound Zn. Samples at 0.5 mg/ml protein were dissolved in 2% nitric acid prior to analysis.

Laminin Polymerization Assay

Polymerization was assayed, in vitro, as described in (17) . Laminin (0.3 mg/ml), initially centrifuged to remove aggregates, was incubated for 4 h at 37 °C, under 4 different conditions: TBS + 2 mM EDTA, TBS + 1 mM CaCl(2), TBS + 15 µM ZnCl(2), or TBS + CaCl(2)/ZnCl(2). Samples were then centrifuged for 15 min, at 12,000 times g, and the polymerized fraction was calculated by subtracting the supernatant concentration from the total.


RESULTS

Purification of Laminin and Entactin

Laminin and entactin were purified by NaCl extraction (see ``Materials and Methods'') and gel filtration chromatography. The laminin-entactin complex (normally in a 1:1 stoichiometry) was isolated from the EHS extract by gel filtration on a Bio-Gel A-5m column eluted with TBS (Fig. 1A). Dialysis against TBS/2 M guanidine HCl dissociated the complex, and laminin and entactin were separated by gel filtration on a Sephacryl-S400HR column (Fig. 1B). Urea (2 M) did not completely dissociate the complex (not shown). Nonlathyritic mice were used to maintain collagen type IV cross-linking and minimize contamination of the laminin. Trace amounts of collagen type IV, possibly complexed with laminin, were resolved in the leading minor peak (Fig. 1B), as determined by polyacrylamide gel electrophoresis (not shown). A yield of 1.96 ± 0.28 mg of laminin and 0.21 ± 0.03 mg of entactin per g of tumor (n = 4) was achieved which is comparable to the yields attained with the EDTA extraction method(25) .


Figure 1: Purification of laminin and entactin. A, isolation of the laminin-entactin complex by gel filtration on Bio-Gel A-5m column eluted with TBS at 25 ml/h. Inset is an SDS-polyacrylamide gel electrophoresis (5-10%) showing EHS homogenate, H; laminin-entactin complex, L:E; laminin, L; and entactin, E. B, separation of the dissociated components by gel filtration on Sephacryl-S400 HR column eluted with TBS/2 M guanidine HCl at 25 ml/h.



Laminin Binding to Entactin

Laminin binding to entactin and collagen type IV was investigated by enzyme-linked immunosorbent assay under various conditions to better understand the biochemical nature of these interactions. Binding experiments (Fig. 2) revealed that laminin-entactin interactions occurred with an affinity of K(d) = 1.5 ± 0.6 nM, B(max) = 33.8 ± 5.2 ng (n = 3), which is in agreement with the published values of 1-10 nM for EHS entactin (27) and K(d) leq 1.0 nM for recombinant entactin(19) . The binding data fit one class of binding site model best supporting the conclusion made by others, that there is only one entactin binding site on laminin. But positive confirmation was not possible with this assay since neither the orientation nor the availability of binding sites could be determined.


Figure 2: Effectors of laminin-entactin binding. Entactin was coated (20 ng/well) onto microtiter plates and incubated with increasing concentrations of laminin in the presence of different compounds, as shown on the right of the graph. Symbols and lines represent experimental and computer-generated theoretical values of bound laminin, respectively. Control is buffer + 15 µM ZnCl(2) showing a binding activity of K = 1.8 nM, B(max) = 38.2 ng. All the other binding conditions shown, except EDTA, also included 15 µM ZnCl(2).



The protein denaturant urea at 2 M prevented binding, indicating the interaction was conformation-dependent (Fig. 2). The reduction in binding when the NaCl concentration was increased (0.3 M) indicated that the interaction was also ionic in nature. Free sulfhydryl groups were also implicated by the reduction in binding activity observed after alkylation with N-ethylmaleimide without reduction of disulfide bonds. Also, the inhibition of laminin binding activity with EDTA suggested a divalent metal requirement, not previously observed, and, of a battery of common trace elements tested at their respective normal plasma concentrations, zinc was the most effective at enhancing laminin binding activity (Fig. 3). Furthermore, the effect zinc had was exerted through laminin only since preincubation of the entactin with Zn was no more effective at increasing binding activity than omitting Zn altogether. CuCl(2) was the only other divalent ion that had a measurable effect on laminin binding activity, probably reflecting its similarity in atomic mass to Zn (Cu = 63.55 versus Zn = 65.38). The binding maximum with Cu was much lower than that observed with Zn. Trivalent metals such as Fe and Al were found to cause a nonspecific increase in laminin binding activity and are likely not involved in this interaction (not shown). The suppression of binding by heparin may be caused by steric hindrance, although the heparin binding regions on laminin that have been mapped (VI domain of alpha short arm and G domain of alpha1 long arm) appear to be clear of the entactin binding domain(28, 29) . Alternatively, the inhibitory effect of heparin may be due to the sequestration of Zn(30) .


Figure 3: Metals and laminin-entactin binding. The influence of different divalent metals on laminin-entactin binding was evaluated (2 mM CaCl(2), 2.7 nM CdCl(2), 15 µM CuCl(2), 1 mM MgCl(2), 11 nM MnCl(2), 15 µM ZnCl(2)). Similar results (little or no binding) were obtained with CaCl(2), CdCl(2), MgCl(2), and MnCl(2) and are represented by one curve for simplicity. Dissociation constants and binding maximums are shown on the graph.



The zinc effect on laminin-entactin binding activity was saturable with optimal binding occurring at physiological Zn concentration (15 µM) (Fig. 4). However, as the zinc concentration was raised above 15 µM, the amount of nonspecific binding increased (Fig. 4B), suggesting the influence of Zn on specific binding activity was saturated at 15 µM. Laminin (0.5 mg/ml) dialyzed against an excess of ZnCl(2) (50 µM, 1 liter) followed by TBS to remove free metal, was found to contain 8.4 ± 1.7 mol of Zn/mol of laminin (Fig. 4C). A small amount of Zn, 1.3 ± 0.8 mol of Zn/mol of laminin, was also detected for laminin dialyzed against TBS only. Incubation at higher ZnCl(2) concentrations (>50 µM) was avoided since it caused the, albeit reversible, precipitation of laminin.


Figure 4: Zinc effect was saturable. A, laminin-entactin binding was investigated with [ZnCl(2)] increasing from 0 to 50 µM. B, nonspecific binding activity (laminin-bovine serum albumin) was investigated over the same range of [ZnCl(2)] and, above 15 µM ZnCl(2), increased significantly (open symbols). C, laminin-bound zinc, at 1.3 ± 0.8 mol/mol for untreated laminin (unloaded) and 8.4 ± 1.7 mol/mol for laminin incubated with 50 µM ZnCl(2) (loaded), was detected by flame atomic absorption spectroscopy. Values are based on the mean and range for two separate laminin preparations.



Inspection of the laminin amino acid sequence revealed 12 cysteine-rich repeats which contain nested zinc finger consensus sequences not previously reported (Fig. 5). Furthermore, the entactin binding site was recently mapped to a zinc finger-containing Cys-rich repeat on the laminin 1 chain(1) .


Figure 5: Alignment of laminin Cys-rich repeats containing nested zinc finger consensus sequences. Of 42 Cys-rich repeats in the laminin alpha1, beta1, and 1 chains, 14 are aligned with the zinc finger consensus sequence; the shaded area highlights putative zinc finger motifs, the mitogenic peptides RGD and YIGSR are underlined, and the asterisk (*) marks the repeat containing the entactin binding site(1) . C, cysteine; a, cysteine or histidine; and x, any amino acid.



Laminin Binding to Collagen Type IV

Laminin did not bind collagen type IV (Collaborative Research) in the absence of divalent metals (2 mM EDTA) (Fig. 6). The addition of CaCl(2) had little effect on the binding. However, with the addition of 15 µM ZnCl(2), a high affinity interaction was detected, involving a single class of binding sites, with an affinity of K(d) = 5.4 nM, B(max) = 39.0 ng. At higher NaCl concentrations, this binding was reduced, indicating that there was an ionic component to the interaction. Alkylation with N-ethylmaleimide, without reduction of the disulfide bonds, decreased the binding affinity (K(d) = 11.7 nM) suggesting free sulfhydryls could be important for the binding site. When entactin was included, at a 2 M excess of laminin, the affinity of the laminin-collagen type IV interaction was increased but with a reduced B(max) (K(d) = 2.4 nM, B(max) = 15.0 ng). This binding characteristic most likely reflects the preferential formation of laminin-entactin-collagen type IV ternary complexes. The affinities are within the ranges previously reported for entactin-collagen type IV (K(d) = 2-10) and for laminin-entactin-collagen type IV (K(d) = 9-20 nM)(18) . Recombinant entactin bound collagen type IV and laminin with a higher affinity (K(d) leq 1.0 nM)(19, 34) .


Figure 6: Laminin-collagen type IV binding was influenced by Zn and entactin. Collagen type IV was coated (100 ng/well) onto microtiter plates and incubated with increasing concentrations of laminin with either no metal, 2 mM CaCl(2), 15 µM ZnCl(2), 0.3 M NaCl + 15 µM ZnCl(2), 10 mMN-ethylmaleimide (NEM) + 15 µM ZnCl(2), or entactin (2 M excess to laminin) + 15 µM ZnCl(2). Dissociation constants and binding maximums are shown on the graph.



Laminin Polymerization Is Independent of Zn

The polymerization of laminin has been found to be dependent on calcium ions (35) and could not be substituted with Zn (Fig. 7). When laminin (0.3 mg/ml) was incubated in TBS + 1 mM CaCl(2), 40-45% of the monomer polymerized. ZnCl(2) could not replace CaCl(2), and its effect on polymerization was not significantly different from that observed with EDTA (Student's t-test, p < 0.05). Furthermore, ZnCl(2) did not interfere with CaCl(2) mediation of the process, since incubation with CaCl(2), or with CaCl(2) + ZnCl(2), showed no difference in polymerization (Student's t-test, p < 0.05). This is consistent with the lack of putative Zn finger sequences in the domains involved in polymerization.


Figure 7: Laminin polymerization proceeded independent of zinc. Polymerization was assayed based on the method in (17) . Laminin at 0.3 mg/ml was incubated under the four different conditions shown at 37 °C for 4 h, centrifuged for 15 min at 12,000 times g, and the polymer fraction was calculated by subtracting the supernatant concentration from the total. Percent laminin polymerized was plotted, based on the mean and standard deviation of 3 experiments. Analysis of the data by Student's t test indicated that EDTA versus ZnCl(2) and CaCl(2)versus ZnCl(2)/CaCl(2) (p < 0.05) were not significantly different.




DISCUSSION

Basement membrane formation involves the secretion of a small set of high molecular weight proteins/proteoglycan which spontaneously interact to generate a supermolecular matrix. The identification and characterization of the specific binding sites directing this assembly is an area under active study. Its been proposed that the ``backbone'' for BM consists of two independent polymers of laminin and collagen type IV which become interconnected by either direct laminin-collagen type IV associations (20, 21) or by the actions of a bridging protein, entactin(18) .

The organization of BM is not uniform, and both developmental and tissue-specific heterogeneity in its structure is likely influenced by the expression of different laminin and collagen type IV isoforms(3, 36) . Biochemical factors, however, may also contribute to the final structure and function of BMs, and our data suggest Zn may be added to the short list of putative effectors governing BM organization. These include phospholipid, Ca, and heparin. The critical laminin concentration required for polymerization is lower on lipid bilayer surfaces such as plasma membranes (37) and may explain why BM form in close proximity to the cells which synthesize it. At physiological concentrations, both Ca(35) and heparin (28) promote laminin polymerization. Conversely, heparin is an inhibitor of collagen type IV polymerization(38) . We have shown that both heparin and Ca block laminin-entactin binding, and, in light of their effects on the polymerization process, they may serve to reduce the cross-linking of the laminin-collagen type IV networks favoring the formation of laminin-rich BM.

The positive effect of zinc on laminin binding activity suggests that it could be a potential metal co-factor for BM assembly and organization. Preincubating entactin or collagen type IV with ZnCl(2) did not enhance laminin binding activity, indicating Zn was affecting laminin only. However, entactin has been shown previously to bind both zinc- and cobalt-loaded columns equally well before and after complete alkylation of the protein(34) . This indicated that metal binding was not dependent on protein conformation and likely occurred via the His-Xaa-His sites, which are known to bind certain metals with high affinity(57) . In addition, Reinhardt et al.(34) reported that treating entactin with 2 mM EDTA had little effect on its binding to either the laminin P1 pepsin proteolytic fragment or collagen type IV, consistent with our proposal of the role of Zn as a laminin-specific co-factor. They also found that Zn at 50 µM inhibited binding of entactin which again is in agreement with our observation that zinc concentrations above physiological (15 µM) interfered with laminin-entactin interactions by increasing nonspecific binding. Above 50 µM ZnCl, laminin precipitated in solution.

Laminin-collagen type IV binding was enhanced by zinc generating a binding maxima which was significantly higher than for laminin + entactin-collagen type IV, indicating that entactin redirected laminin binding to a smaller number of binding sites. The apparent increase in affinity for the laminin-collagen type IV interactions, when entactin was included in the assay, was likely caused by the formation of stable ternary complexes(19) . The number of binding sites to which laminin was limited to was about one-third that seen with Zn alone possibly because of steric blocking by entactin of the collagen type IV binding sites on the laminin alpha1 and 1 chains. Aumailley et al.(18) also found that laminin-entactin complex bound collagen type IV with high affinity while little or no binding was detected with isolated laminin, as we observed in the absence of zinc.

Laminin binding sites have been mapped to sites approximately 80 nm, 200 nm, and 300 nm distal of the carboxyl-terminal globular domain of collagen type IV(21) , while the entactin binding sites were located at the first two sites only(18) . Our binding assay data suggest that at least 2 types of mutually exclusive interactions cross-linking the laminin and collagen type IV networks are possible; one that is solely Zn-mediated and the other of higher affinity involving Zn + entactin as the bridging molecule.

When we investigated the laminin sequence for potential Zn binding regions structural motifs rich in cysteine residues caught our interest. These motifs of unknown function are common for basement membrane components. Two types have been recognized, based primarily on the number and spacing of the cysteine residues within a 40-amino acid or 60-amino acid domain (Fig. 8). One type has 6 Cys with a spacing similar to the active domain of the pancreatic secretory trypsin inhibitors of the Kazal family(39) . The other more common type, also with 6 Cys, is similar to the Cys-rich domains in epidermal growth factor (EGF)(46) . This domain aligns best with the protein products from the Drosophila melanogaster neurogenic locus Notch (41) and the Lin-12 protein from Caenorhabditis elegans(47) . Also included are other proteins which have fewer copies of the repeat such as the low density lipoprotein receptor(48) , factor IX, factor X, and protein C(49, 50) , and plasminogen activator(51) .


Figure 8: Alignment and comparison of Cys-rich repeats common for BM components with that for pancreatic secretory trypsin inhibitors (PSTI) and epidermal growth factor (EGF), showing Cys number and spacing consensus. These included PSTI(39) , EGF(40) , Notch(41) , entactin/nidogen(27) , BM-90/fibulin(42) , SPARC/osteonectin(43) , agrin(44) , laminin (31, 32, 33) , and perlecan(45) . Cysteine spacing similar to PSTI is found in SPARC and to EGF in BM-90, agrin, entactin, and also SPARC. C, cysteine; X, any residue.



The inner rod-like regions of the laminin short arms also contain many Cys repeats which have mitogenic activity for a variety of cell types, that is independent of the EGF receptor(52, 53) . The nonapeptide GDPGYIGSR and the shorter pentapeptide YIGSR are both found in Cys-rich repeats in domain III of the 1 chain and have been shown to promote cell attachment and migration(54, 55) .

On closer examination of these Cys-rich repeats, both laminin and perlecan contain 60-amino acid repeats of 8 Cys exclusively that exhibit much lower similarity (particularly in Cys spacing) to the EGF-like repeat and may constitute a third type of repeat (Fig. 8). Within the 42 Cys-rich repeats found on laminin, 12 appear to harbor the consensus sequence for Cys-rich zinc fingers, a proven zinc binding domain in many systems (Fig. 5). Based on our data and the aforementioned sequence information, we propose that as many as 12 zinc finger-containing repeats on laminin could potentially be coordinated by Zn atoms. These may generate one high affinity entactin binding site and possibly one or more collagen type IV binding sites.

It is unknown if disulfide bonding takes place in all of the Cys repeats of laminin and perlecan, but a hypothetical disulfide bonding pattern involving all 8 Cys has been proposed by Appella et al.(46) and Engel(52) . However, their proposal is based on the secondary structure for an EGF repeat from human pro-EGF(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48) determined by NMR which agrees with a predicted disulfide bonding pattern between all 6 Cys (1-3, 2-4, and 5-6)(56) . In laminin, only 5 of 8 Cys in the repeats align with those of the EGF domain(31, 46) . It is possible that in the absence of Zn free sulfhydryls could form disulfide bonds but our data suggests that the Cys-4, -5, -6, and -7 in at least 8 of 42 repeats may be coordinated by Zn generating a zinc finger-like motif. High resolution studies to determine the conformation of these Cys repeats in the absence or presence of Zn is needed.

Laminin isolated from EHS tumor was found to have about 1 mol of Zn/mol of laminin which, after incubation with 50 µM ZnCl(2), could be increased to about 8 mol/mol of laminin, an amount consistent with the predicted number of zinc finger sequences. In further support of this idea, the entactin binding site was recently mapped to a cysteine-rich repeat on the laminin 1 chain(1) , which happens to contain a zinc finger-like sequence. Three out of twelve Cys-rich repeats on this chain have nested zinc finger consensus sequences. Mayer et al.(1) observed that iodination of the B2III-4 peptide (58-mer), which contained 2 Tyr residues in the nested zinc finger sequence, substantially reduced binding activity. Furthermore, reduction and alkylation of the Cys residues abolished its ability to inhibit laminin-entactin binding. We have confirmed that alkylation of Cys reduced laminin binding activity for both entactin and collagen type IV. Laminin polymerization was found to be Ca-dependent as reported elsewhere and could not be reproduced with Zn, nor did Zn interfere with the effect of Ca, of which 2-3 are required to bind to the terminal globular domain of 1 chain to facilitate maximal laminin polymerization(35) . Most likely, the two ion species bind to different sites on laminin consistent with the lack of zinc finger-like sequences in the terminal globular domains required for polymerization. Hence, laminin zinc fingers may function mainly in the lateral associations interconnecting the laminin and collagen type IV networks. One or more may also be involved in the mitogenic activity of laminin directly, or indirectly, by influencing the accessibility of the RGD or YIGSR peptides found on 2 different Cys-rich domains between zinc finger-containing repeats.

These putative zinc finger motifs on laminin are highly conserved between human(58, 59, 60) , mouse(31, 32, 33) , and Drosophila(61, 62, 63) . For mouse and human, the number and relative location of the zinc finger sequences are identical. In Drosophila, the most distantly related species examined, 7 out of 9 of its zinc finger sequences, including the one at the entactin binding site, have maintained their relative locations in the laminin sequence, with an amino acid sequence identity of about 60% between Drosophila and mammals. Perlecan (mouse), a BM proteoglycan, also contains 6 Cys-rich repeats(45) , of which 3 contain zinc finger-like sequences (Fig. 9). Overall sequence similarity between laminin alpha1 chain and perlecan is high, particularly at the amino end suggesting a common evolutionary origin (64, 65) . Perlecan has also been reported to bind entactin(66) , but whether any of the zinc finger-containing repeats act as the entactin binding site remains to be established.


Figure 9: Perlecan Cys-rich repeats (45) containing zinc finger consensus sequences. 3 out of 6 Cys-rich repeats which contain zinc finger sequences (shaded) are aligned.




FOOTNOTES

*
This work was supported by Medical Research Council of Canada Grant MT-3153. 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.

§
To whom correspondence should be addressed. Tel.: 613-548-6048; Fax: 613-545-2907.

(^1)
The abbreviations used are: BM, basement membrane; EGF, epidermal growth factor.

(^2)
H. Kleinman, personal communication.


ACKNOWLEDGEMENTS

We thank Dr. J. S. Poland and M. K. Andrews, analytical services, Dept. of Chemistry, Queen's University, for technical assistance with the atomic absorption spectroscopic analysis.


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