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INTRODUCTION |
Laminins are a growing family of large extracellular glycoproteins
found in but not confined to basement membranes (1-3). These
heterotrimeric proteins are composed of one
, one
, and one
type of chain that assemble together through a triple-helical coiled
coil motif (4). Today, 10 different laminin chains have been
characterized from mammals (5-18) that theoretically could give rise
to over 100 different isoforms. This extreme variability reflects the
specialization of basement membranes in mature tissues (19, 20) as well
as during development (21, 22). Moreover, the heterogeneity in
composition provides us a concrete, molecular level handle as to how
basement membranes influence cell behavior and differentiation in a
given tissue. The characteristic and often delimited symptoms in the
various laminin gene defects (23-29) further emphasize the biological
significance of the variation within the laminin family.
There are two laminin
chains established to date (7, 8, 12). The
2 chain is expressed restrictedly and is mainly confined to
epithelial cells (12, 30). Of the eleven characterized isoforms, only
laminin-5 (
3
3
2) contains the
2 chain (31-33). In contrast,
the
1 chain is widely expressed (12, 22, 34, 35) and takes part in
all the other known isoforms (36-41). This dominant position differs
from the situation among the
and
chains, both classes having at
least one widely expressed, alternative chain (22, 38, 42-45). We,
therefore, considered the possibility of the existence of a third
chain. Such a chain could be particularly interesting with respect to
the size of the laminin family. An alternative
chain would nearly
double the number of potential laminin isoforms and, more importantly,
proportionally increase the number of identifiable components embedded
in the protein meshwork in basement membranes. Clearly, this would
significantly improve the resolution of our current basement membrane
models that are based on a limited number of distinct proteins
(46-49).
In a primary sequence, a coiled coil motif is seen as a seven-amino
acid-long repeat (a, b, c, d, e, f, g) in which the residues at
positions a and d are predominantly hydrophobic (50, 51). Once the
coiled coil is formed, these residues are effectively protected from
the polar environment of tissue fluids. While the hydrophobic
interactions greatly favor the coiled coil assembly, the specificity of
the interaction is often determined by the polar residues running
parallel on either side of the hydrophobic core as well as by the polar
and charged residues embedded in the core (52, 53).
In this study, we predicted the hydrophobic profile of a
chain core
in the C-terminal part and used this profile to discriminate against
other coiled coil sequences that we found from the translated EST1 data base at National
Center for Biotechnology Information (54). Using this strategy, we
could identify and further characterize a novel laminin
3 chain.
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MATERIALS AND METHODS |
Data Base Search--
The EST data base at the National Center
for Biotechnology Information (http://www.ncbi.nlm.nih.gov) was
searched with tblastn sequence comparison program (55) installed at the
National Center for Biotechnology Information server. A C-terminal
sequence from the human laminin
1 chain (8) was used as a query. The
probability of finding a match by chance was set to E = 1000, since the sequence homology between laminin chains in the coiled
coil regions is usually low, typically about 20-30% identical
residues (10, 16-18). Previously uncharacterized translations matching
the query were first selected for the presence of a cysteine within the 10 C-terminal residues. To compare the amphipathicity and the hydrophobic profile along the hydrophobic core, the remaining translations were analyzed with the helical wheel program from the
Genetics Computer Group software package (56). This permits easy
visualization of the aligned residues in a and d positions of
successive turns of the helix. A murine EST clone (373279) from the
WashU-HHMI Mouse EST Project at the Washington University (57) was
found to have a similar hydrophobic profile to the laminin
1 and to
the
2 chains. Furthermore, the sequence of this clone
(GenBankTM accession number W64443) was found to be similar
to that of the laminin
1 chain by the WashU-HHMI Mouse EST Project.
Clone 373279 was purchased from Genome Systems and used as a probe for Northern analysis and for screening a mouse testis-specific cDNA library (CLONTECH).
Molecular Cloning--
Two methods were used for obtaining
additional clones from the same gene as the 373279 clone. First, a
mouse testis-specific cDNA library was screened with gene-specific
restriction fragments (58). Second, various gene-specific primers and a
custom anchor primer 5'-tcgagcggcctcccgggcaggt-3' were used in rapid
amplification of cDNA ends for amplifying flanking fragments from
embryonic mouse cDNA (E15, CLONTECH). For
sequencing, the cDNA fragments were ligated into pBluescript or
pCRScript vectors (Stratagene). The cycle sequencing reactions were
done with Taq-derived polymerase using termination chemistry
(Perkin-Elmer) and the extension products resolved with Applied
Biosystems 377 and Applied Biosystems 310 automated sequencers. All the
oligonucleotides used in this study were synthesized with Applied
Biosystems 392 DNA/RNA oligosynthesizer. The sequence data obtained
were analyzed with the Genetics Computer Group software (56).
Northern and in Situ Hybridization Analyses--
An RNA blot
with 2 µg/lane of poly(A)-RNA from various adult mouse tissues
(CLONTECH) was hybridized according to standard procedures (58). Two probes giving essentially identical results were
sequentially applied: a 0.7-kb fragment from the coiled coid region
(nucleotides 3585-4263) and a 0.45-kb segment from the 3'-UTR. Random
priming strategy was applied in labeling the probes (Prime-a-gene,
Promega). The filters were hybridized at +42 °C for 16-24 h in a
solution containing 50% formamide, 2% SDS, 50 µg/ml of
polyvinylpyrrolidone (Pharmacia), 50 µg/ml bovine serum albumin, 50 µg/ml Ficoll 400 (Pharmacia), and 5 × SSPE (750 mM NaCl, 50 mM sodium phosphate, pH 7.4, 5 mM
EDTA). After washing in 1 × SSC + 0.1% SDS for 2 × 15 min
and in 0.1 × SSC + 0.1% SDS for 15 min, Kodak X-Omat films were
exposed to the filter-bound radioactivity for several days at
70 °C using intensifying screens.
In situ hybridization was carried out on tissues from adult
mouse essentially as described elsewhere (44, 59, 60). Briefly, tissues
were fixed in the fresh 4% paraformaldehyde, dehydrated by a gradient
of ethanol, embedded in paraffin, and sectioned. Following postfixation
in 4% paraformaldehyde, the sections were incubated in
phosphate-buffered saline containing 0.1% active diethyl pyrocarbonate
(Sigma), equilibrated in 5 × SSC and prehybridized for 2 h
at 55 °C. Hybridization was then carried out overnight at 55 °C.
For generation of single-stranded RNA probes, a 450-base pair
PstI fragment from the 3'-UTR of the cDNA was cloned
into pGEM3Z (Promega). Antisense and sense riboprobes were labeled with
digoxigenin-11-UTP by in vitro transcription with T7 and SP6
RNA polymerase (Roche Molecular Biochemicals), respectively. After
washing in 50% formamide and standard sodium citrate, the sections
were incubated with an alkaline phosphatase coupled anti-digoxigenin antibody and stained using nitro blue tetrazolium and
5-bromo-4-chloro-3-indolyl phosphate solutions (Roche Molecular Biochemicals).
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RESULTS AND DISCUSSION |
Molecular Cloning--
The specificity of laminin chain assembly
lies at the coiled coil domain. According to our hypothesis, this is
reflected in the chain-specific hydrophobic profiles in the coiled coil
core. Similarly, the intraheptad position of the C-terminal cysteine residues in the coiled coil domain is chain-specific. Starting from
these premises, the EST data base at the National Center for
Biotechnology Information was searched as described under "Materials
and Methods." The EST clone 373279 from the mouse EST project at the
Washington University (see "Materials and Methods") fulfilled these
criteria. Furthermore, the sequence of this clone was found to be
significantly similar to that of the murine
1 chain by the EST
project. We then used the clone 373279 as a starting point in the
search for overlapping clones. Together with 373279, the identified
clones encoded for an open reading frame of 4829 nucleotides that
included 53 base pairs of tentative 5'-UTR (Fig. 1).

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Fig. 1.
Schematic presentation of the murine
laminin 3 cDNA. Top, scale
in base pairs. Bottom: cDNA clones isolated in this
study together with the EST clone 373279. Closed arrow = translation initiation codon; open arrow = translation termination codon.
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Sequence Comparison with Other Laminin
Chains--
Further
analysis of the cDNA-derived peptide sequence predicts a 33-amino
acid-long signal peptide (61, 62) and a 1559-amino acid-long mature
peptide showing a prototype domain structure of a laminin
chain
(Figs. 2 and
3). Comparison with the human
1 chain
reveals 64.0, 62.1, 32.5, 54.3, and 22.0% amino acid identity in
domains VI, V, IV, III, and II/I, respectively (8). The corresponding
analysis with the human
2 chain (12) yields 51.5, 34.4, 47.1, and
22.2% for domains V, IV, III, and II/I. Therefore, we name this new
chain as laminin
3 chain in agreement with the established
nomenclature (3).

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Fig. 2.
Comparison of the amino acid sequences of the
murine laminin 3 and
1 chains. Alignment was produced with the
fasta program in the GCG package (56). The first letter of Roman
numbers indicates the start of domains. mlamg3 = murine
laminin 3 chain; hlamg1 = human laminin 1 chain
(8).
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Fig. 3.
Comparison of the domain structure of
laminin chains. The sequence-derived
domain structures of laminin chains are illustrated. N termini are
shown on the left. LN, NE, L4, and coiled coil modules (71)
are indicated. The corresponding LE and L4 modules are
shaded. 1, 2,
3 = 1, 2, and 3 chains, respectively.
Inset, the fourth LE module in domain III (4) of the murine
3 chain. The five key residues involved in nidogen binding in the
murine 1 chain (66, 70) are all conserved and the corresponding
residues in the 3 chain are shaded. Cysteine bridges are indicated
with thick lines. Display design was adopted from (70).
a, b, c, d = the four
loops in LE modules.
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Using the
1 chain as a model, some predictions about the properties
of the
3 chain can be made. First, it is likely that the dimensions
of the two chains are similar. The lengths of the coiled coil domain
are 579 (
1) and 569 (
3) residues. The globular domains VI and IV
are of similar size as well. Furthermore, both chains have 11 copies of
laminin repeat motifs (63, 64). Second, the domains to which specific
functions have been mapped in the
1-chain appear to be conserved:
domain VI with a calcium-dependent laminin self-assembly
activity (49, 65) and nidogen binding site in the fourth laminin repeat
in the III domain (66) (see Fig. 3). Taken together, this suggests that
the
3 chain possesses a full networking capability similar to the
1, but different from the
2 chain.
Expression of the Laminin
3 Chain--
To investigate the
spatial expression in mouse tissues, an RNA blot containing RNA from
various adult organs (CLONTECH) was hybridized
serially with two laminin
3 chain specific probes (Fig.
4). The strongest signals were obtained
from testis, kidney, and lung, with low level of expression in brain,
skeletal, and heart muscle and intestines. Both probes revealed two
transcripts of about 6 and 8 kb. The source for the different mRNAs
remains unknown. 3'-rapid amplification of cDNA ends using a primer
at position 4748-4771 of the full-length laminin
3 chain cDNA
yielded an about 1.3-kb-long product (not shown). In the case of the
1 chain, the 3'-UTR contains several polyadenylation signals that probably are alternatively used (67) and thus account for the two
mRNAs observed (8).

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Fig. 4.
Northern analysis of expression pattern of
the laminin 3 chain in adult mouse
tissues. RNA size markers on the left are: 9.5, 7.5, 4.4, 2.4, and 1.35 kb. H = heart; B = brain; S = spleen; L = lung;
Li = liver; M = skeletal muscle;
K = kidney; T = testis.
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To further characterize the tissue compartments expressing the
3
chain, in situ hybridization experiments were performed on
adult kidney and testis tissues. In the kidney, strong signals were
seen in either afferent or efferent arterioles of the glomeruli (Fig.
5A), and in endothelial cells of other
arteries (Fig. 5, B and C), while cells of the
actual glomeruli only showed modest staining above background (Fig. 5,
A and B). Tubular or interstitial cells did not
exhibit signals, but capillaries located between tubular structures
were clearly positive for expression (Fig. 5D). In the
testis, the strongest signals were seen in the Leydig cells located
between the seminiferous tubuli (Fig. 6,
A-C). The sertoli and spermatogenic cells were negative.
Staining with the sense probe did not yield any distinct
staining (Fig. 6D).

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Fig. 5.
Expression of laminin
3 chain in adult kidney. A,
staining is seen in a blood vessel identified as either an afferent or
efferent arteriole (arrow). G, glomerulus.
B, a strong staining is observed in an arteriole adjacent to
a glomerulus (arrow). Weak signals are seen in some
glomerular cells (small arrow). C and
D, strong staining is also observed in a medium size artery
of the cortex (C), as well as in capillaries located in between renal
tubuli (arrows in D). E, negative
control using a sense probe.
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Fig. 6.
Expression of the laminin
3 chain in adult mouse testis as determined by
in situ hybridization. A, staining
with the antisense probe is seen between the seminiferous tubules
(arrow). Sertoli or spermatogenic cells are negative.
S, seminiferous tubule. B, schematic illustration
of the section in A, showing several seminiferous tubuli (S)
containing Sertoli and spermatogenic cells located in the tubules above
the basement membrane. Leydig cells are located in the interstitial
space filling the interstitium between adjacent tubules (dark
pink areas). C, magnification of the box in
A shows that the expression is present in Leydig cells.
D, staining with the negative control sense probe.
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The expression pattern of the laminin
3 chain overlaps with that of
the other
chains. In kidney, several cell types express the
1
chain (12, 68). The
2 chain expression is confined to the epithelial
cells (12, 68). In testis, the laminin
1 chain is found in the
seminiferous tubules (69). However, little is known about the
expression of laminin
chains by Leydig cells.
In general, the results of the in situ hybridization
analyses indicate that endothelial cells of arteries and capillaries, as well as interstitial cells in certain tissues, express the
3
chain. However, expression is apparently not significant in all
endothelial cells, as tissues rich in blood vessels and capillaries, such as cardiac and skeletal muscles, do not exhibit strong expression, e.g. as is the case for the laminin
4 chain (22, 44, 45). Further studies are needed to show in detail the expression pattern in
epithelia. The present results indicate that, with the exception of
Leydig cells in the testis, mesenchymal cells are not major producers
of laminin containing the
3 chain.
By the virtue of their coexpression with the
3 chain, the
2,
3,
4, and
5 chains could take part in the assembly of a
3
chain containing laminin (22, 30, 38, 42, 44, 45). Since both the
1
and the
2 chains are also expressed at these sites (34, 35, 38, 42,
43), at least eight hitherto uncharacterized laminin isoforms could
collectively reside in these tissues. These tentative isoforms are
likely to integrate into the basement membrane protein meshwork via the
conserved networking promoting domains VI (49, 65) and III(LE4) (66, 70).