(Received for publication, October 10, 1995)
From the
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.
Basement membrane (BM) ()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 1,
1, 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
1,
1, 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.
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).
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.
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 showing a binding activity
of K
= 1.8 nM, B
= 38.2 ng. All the other binding
conditions shown, except EDTA, also included 15 µM ZnCl
.
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
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
short arm and G
domain of
1 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, 2.7 nM CdCl
, 15 µM CuCl
, 1 mM MgCl
, 11 nM MnCl
, 15 µM ZnCl
). Similar results (little or no binding) were
obtained with CaCl
, CdCl
, MgCl
, and
MnCl
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
(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
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] increasing from 0 to 50
µM. B, nonspecific binding activity
(laminin-bovine serum albumin) was investigated over the same range of
[ZnCl
] and, above 15 µM ZnCl
, 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
(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 1,
1, 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.
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
, 15 µM ZnCl
, 0.3 M NaCl + 15 µM ZnCl
, 10 mMN-ethylmaleimide (NEM) + 15 µM ZnCl
, or entactin (2 M excess to laminin)
+ 15 µM ZnCl
. Dissociation constants and
binding maximums are shown on the graph.
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 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
and CaCl
versus ZnCl
/CaCl
(p < 0.05) were not significantly
different.
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 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
1
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
, 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 1 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.