(Received for publication, September 27, 1995)
From the
We have identified a fifth member of the subfamily of
vertebrate laminin chains. Sequence analysis revealed a close
relationship of
5 to the only known Drosophila
chain, suggesting that the ancestral
gene was more similar to
5 than to
1-4. Analysis of RNA expression showed that
5 is widely expressed in adult tissues, with highest levels in
lung, heart, and kidney. Our results suggest that
5 may be a major
laminin chain of adult basal laminae.
Laminins are major glycoproteins of basal laminae throughout the
vertebrate body. Originally identified as structural components, it is
now clear that laminins are also signaling molecules that regulate the
proliferation, motility, and differentiation of the cells they contact (1, 2, 3) . The first laminin
discovered(4, 5) , now called laminin-1(6) ,
is a trimer of related A, B1, and B2
chains(7, 8, 9) . The subsequent discovery of
the novel laminin chains S-laminin (10) and merosin-M (11) revealed that the laminins comprised a larger gene family
than initially envisioned. More recently, four additional chains have
been
cloned(12, 13, 14, 15, 16, 17, 18, 19) .
So far, however, all laminin chains sequenced resemble either A, B1, or
B2, and all of the laminins purified are trimers containing an A-like,
a B1-like, and a B2-like
chain(6, 19, 20, 21, 22) .
Based on these findings, a new nomenclature has been adopted in which
laminin chains are divided into (A-like),
(B1-like) and
(B2-like) subfamilies; A, M, B1, S, and B2 are now called
1,
2,
1,
2, and
1, respectively(6) .
Consistent with laminin's trimeric structure, all basal
laminae characterized to date contain at least one and at least
one
chain(19, 20, 23, 24, 25, 26, 27, 28) .
For the
chains, on the other hand, the situation is less clear.
For example, perineurial basal lamina in peripheral nerve stained
poorly with anti-
1 and not at all with anti-
2(23) .
Likewise, in kidney, glomerular basement membrane was
2-negative
and reacted only moderately well (in human(23) ) or not at all
(in mouse(29, 30) ) with anti-
1. If all laminins
are
/
/
trimers, these results imply that additional
laminin
chains exist. Indeed, several biochemical studies have
provided evidence for an
-like laminin chain distinct from
1
and
2 (31, 32, 33) . The recent
discoveries of the
3 and
4
chains(15, 16, 17, 18) are
provocative in this context, but we (
)and others (16, 18) found little
3 or
4 in several
tissues with
1- and
2-negative basal laminae.
Accordingly,
we undertook a search for additional laminin chains. Using the
polymerase chain reaction, we have identified laminin
5, a novel
murine
chain. Sequence analysis reveals that
5 is more
similar in domain structure to Drosophila laminin
A(34, 35) than it is to mammalian
1-4; the
ancestral vertebrate
chain may, therefore, have been more similar
to
5 that to
1-4. RNA analysis demonstrates that
5
is widely expressed in adult tissues and thus may be a major laminin
chain of adult basal laminae.
Degenerate primers were designed based on sequences conserved
between mouse laminin 1 (7) and human laminin
2(36) . Reverse transcription-PCR (
)was
performed on RNA from postnatal day 7 mouse kidney using a GeneAmp kit
(Perkin-Elmer). One pair of primers from the amino terminus of domain V
(sense, 5`-GGNAGTTGYATHTGYTAYGGNC-3` and antisense,
5`-TRCANTGYTCRCARTTDATNCC-3`, where N = A/G/C/T, H =
A/T/C, and D = G/A/T) produced a fragment of appropriate length
(
330 base pairs). The band was excised from agarose (SeaPlaque
GTG, FMC Bioproducts, Rockland, ME), reamplified with the same primers,
purified with a Wizard PCR Preps kit (Promega Corp., Madison, WI), and
incubated with BglI to digest laminin
1 products, thereby
preventing their further amplification. The remaining full-length
product was reamplified and ligated into the pCRII vector (Invitrogen
Corp., San Diego, CA).
One resulting clone, DB2, bore an insert
related to but distinct from laminins 1 and
2. The DB2 insert
was labeled with [
P]dCTP by the random primed
DNA labeling kit (Boehringer Mannheim) and used to screen an adult
mouse lung oligo(dT)+ random primed
ZAP II cDNA library
(Stratagene, La Jolla, CA). Subsequent cDNA library screening was
performed as a ``walk'' using selected restriction fragments
of hybridizing phage to obtain overlapping clones. Clones were
sequenced on an ABI 373A DNA sequencer using a Taq DyeDeoxy
Terminator cycle sequencing kit (Applied Biosystems Inc., Foster City,
CA). All sequences were determined from both strands. Data base
homology searches were performed on the BLAST server at the National
Center for Biotechnology Information(37) , and sequences were
compared using Genetics Computing Group programs(38) .
For
Northern analysis, a filter containing poly(A)-selected RNA from
several adult mouse tissues (Clontech) was hybridized with a probe
comprising nucleotides 7855-9361 of the 5 sequence. RNase
protection analysis was performed as described previously(24) .
Figure 1:
Amino
acid sequence of the mouse laminin 5 chain, deduced from cDNAs.
Domain boundaries are noted and the adhesive tripeptide sequences, RGD (40) and LRE(42) , are indicated by bullets.
The nucleotide sequence is available from GenBank under accession
number U37501.
The first amino acid
of the deduced sequence is aspartic acid rather than methionine,
indicating that the cDNAs do not reach the 5` end of the coding
sequence. Repeated efforts to obtain additional cDNAs failed. Based on
homology to other laminin chains, we believe that the mature
protein contains
3630 amino acids, of which we have identified
3610.
Figure 2:
Relationship of 5 to other laminin
chains. A, domain structure of the known laminin
chains.
The names of the domains, based on accepted
nomenclature(1, 7) , are to the left of
5.
Percent amino acid identity of individual domains of
5 with the
corresponding domains of the other
chains, determined with the
GAP program(38) , is shown to the left of the other
chains. Numbers of EGF repeats, rounded to the nearest integer, are
indicated within domains III and V. Domain structures of
1 and
1 are shown to indicate the basis for assigning
5 to the
subfamily.
D, Drosophila A. B,
relationships among mammalian and Drosophila
chains,
based on sequence alignment performed by the PILEUP
program(38) . Comparisons were based on domains G-IIIa,
which all
chains contain. C, evolutionary scheme for
vertebrate
chains, incorporating comparisons of primary sequences
(from B) and predicted secondary structure (from A).
In this scheme, the ancestral
gene was more similar in domain
structure to
5 than to
1-4. See text for
details.
Several
features of 5 identify it as an
chain (Fig. 2A). First, it contains a G domain, which is
present in all
but no
or
chains. Second,
5, like
1 and
2, contains three sets of EGF-like repeats (IIIa, IIIb,
and V) and three globular regions (IVa, IVb, and VI) in its
amino-terminal half, whereas no
or
chain has more than two
of each. Third,
5 lacks the
-insert between domains I and II
that characterizes
chains.
Of the four vertebrate laminin
chains characterized to date, two (
1 and
2) contain
domains G-VI, whereas the other two (
3 and
4) are
truncated and contain only domains G-IIIa (Fig. 2A). An alternatively spliced product of the
3 gene,
3B, which contains domains IIIb and IV, has been
identified but not yet fully sequenced(12) . In its domain
structure,
5 is more similar to
1 and
2 than to
3
or
4, and it can therefore be classified as a
``full-length''
chain. On the other hand, within the
domains shared by all
chains, the laminin
5 sequence is more
similar to
3 and
4 than to
1 or
2 (Fig. 2B). Thus, sequence analysis reveals an apparent
discrepancy between relationships based on primary and secondary
structure.
More surprising is that 5 is more similar in domain
structure to the only known Drosophila A chain
(
D(34, 35) ) than it is to any of the vertebrate
chains. Both
5 and
D contain 11 EGF repeats in domain
V, 4 in domain IIIb, and 7 in domain IIIa, whereas
1 and
2
have 4, 9, and 4 repeats in domains V, IIIb, and IIIa, respectively (Fig. 2A). Likewise, domain IVb is much larger in
D and
5 (558 and 577 amino acids) than in
1 or
2
(196 amino acids).
Together, these results suggest the evolutionary
scheme diagrammed in Fig. 2C. We propose than an
ancestral gene, similar in domain structure to
D and
5,
was duplicated early in the vertebrate lineage. One of the daughter
genes evolved the
1/2 domain structure, perhaps by
recombination(39) , and was then duplicated again to generate
1 and
2. The other product of the original duplication was
also reduplicated. One daughter evolved into
5 without further
rearrangement, while the other suffered truncation and then duplicated
yet again to generate
3 and
4. Although speculative, this
scheme accounts for the otherwise puzzling findings that
5
resembles
1 and
2 in secondary structure but is most closely
related to
3 and
4 in primary sequence.
Studies with
synthetic peptides have provided evidence for several discrete adhesive
sites within laminin chains, although their significance in
vivo remains unclear. The tripeptide RGD, which is recognized by
several integrins(40) , is present in
1,
3, and
4 but not in
2 or
D(7, 15, 18, 34, 35, 36, 41) ;
it is present twice within the
5 sequence (Fig. 1). The
tripeptide LRE, a major determinant of a motoneuron-selective adhesive
site in
2(42, 43) , is present in
1,
3,
and
D but not in
2 or
4(7, 15, 18, 34, 35, 36, 41) ;
it is present twice in
5. Finally, the sequence IKVAV, an adhesive
site in
1(44) , is replaced by SKVKV in
5.
Figure 3:
Northern analysis of laminin 5 RNA
from adult mouse tissues. Short (15 h (A)) and long (52 h (B)) exposures of a single blot (with an intensifying screen)
are shown.
The broad
distribution of 5 RNA in adult tissues stands in marked contrast
to the restricted patterns of expression of the
1,
3, and
4 genes(16, 18, 30, 41) . (
)Laminin
2 RNA is widely distributed in adult tissues
but is predominantly expressed by mesenchymal or mesodermal
cells(36) . Taken together, these results suggest that laminin
5 is a major
chain of adult epithelial and/or endothelial
basal laminae. Moreover, it seems possible that reports of
1-like
immunoreactivity in tissues such as kidney, lung, and
muscle(20, 23, 25, 26, 27, 28) ,
which contain little
1 RNA, reflect cross-reactivity of
anti-
1 antibodies with
5. We are currently preparing
monospecific antibodies to evaluate this possibility.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U37501[GenBank].