(Received for publication, May 8, 1995; and in revised form, July 12, 1995)
From the
We have isolated and characterized overlapping cDNA clones
encoding the 3A and
3B chains of mouse laminin 5. Sequence
analysis of the cDNA for the
3B predicts a polypeptide of 2541
amino acids (279,510 Da) comprising a truncated short arm and a
carboxyl-terminal long arm common to the laminin
chains
identified thus far. The short arm of the
3B chain harbors two
alternating epidermal growth factor-like domains and two globular
domains. The amino-terminal globular domain, thought to mediate
interactions with molecules of the extracellular matrix, shows no
significant homology to any globular domain at the tips of the known
laminin isoforms. The
3A cDNA predicts a polypeptide of 1711 amino
acids (186,230 Da) that substitutes a short sequence of 43 amino acids
for the short arm seen in the
3B isoform and displays 77%
conservative homology to the
3Ep chains of the adhesion ligand
epiligrin. Northern and Western blot analyses of skin and lung
epithelial cells demonstrated the tissue-specific expression of the
laminin
3A and
3B isoforms, and in situ hybridization on mouse embryos revealed a focal localization of
3B in areas of the central nervous system.
Laminins are noncollagenous components of basement membranes
that mediate cell adhesion, growth, migration, and differentiation.
These cross-shaped molecules constitute a family of proteins consisting
of three individual polypeptide chains joined together in a long arm as
coiled-coil amphipatic -helices linked by interchain disulfide
bonds. The amino-terminal domain of each of the three chains forms a
distinct short arm (reviewed by Tryggvason, 1993).
Laminin chain
variants with specific patterns of temporal and spatial expression have
been identified in different species. All these isoforms are highly
homologous, in that their short arms are comprised of globular domains
and characteristic epidermal growth factor-like domains, and their long
arms consist of sequences of heptad repeats (Timpl et al.,
1979). On the basis of their primary structure deduced from sequence
data and homology to the polypeptides that compose laminin 1, the
laminin chains characterized thus far have been classified as ,
, or
chains (Burgeson et al., 1994). Only chains
belonging to a different class combine into a trimeric molecule
presenting with a large globular domain G contributed by the carboxyl
terminus of the
chain.
Epithelial cells express specific
laminin isoforms. Laminin 5 was initially identified by a monoclonal
antibody that stains subsets of basement membranes (Verrando et
al., 1987). The protein is associated with the anchoring
filaments, thread-like structures connecting the hemidesmosomes to the
lamina densa of the dermal-epidermal junction (Verrando et
al., 1987; Rousselle et al., 1991). Laminin 5 is composed
of three distinct chains of 165 kDa (3), 140 kDa (
3), and 105
kDa (
2). This mature species derives from a cell-associated
molecule as a result of two extracellular processing events that
generate the
3 and the
2 chains from distinct 200- and
155-kDa precursor polypeptides, respectively (Marinkovich et
al., 1992a). The laminin
3 chain is immunologically related
to a distinct laminin 190-kDa
chain synthesized by keratinocytes
that interacts with a
1 and a
1 chain to form laminin 6.
Laminin 6 and laminin 5 appear to form a complex that functions as a
cell adhesion ligand for integrins
6
4 and
3
1
(Carter et al., 1990; Marinkovich et al., 1992b). In
amnios and fetal skin, the 190-kDa laminin
chain associates also
with a
2 and a
1 chain to form laminin 7 (Wewer et
al., 1994).
Mutations in the genes encoding laminin 5,
including its 3 chain, have been shown to underlie the junctional
forms of epidermolysis bullosa, a recessive inherited skin disorder
characterized by dysadhesion of the epidermis from dermis (Kivirikko et al.(1995) and references therein; Vidal et al.
(1995)).
Screening for cDNA clones from a human keratinocyte
expression cDNA library using a polyclonal antibody against the 3
chain of laminin 5 identified two species of mRNA transcripts.
Partial sequence analysis predicts two polypeptides identical to
the
3
and
3
chain isoforms of the
adhesive ligand epiligrin (Ryan et al., 1994), an anchoring
filament component shown to mediate basal cell adhesion by interacting
with integrin
3
1 in focal adhesion and with integrin
6
4 in hemidesmosome adhesion structures (Carter et
al., 1991). The cDNA encoding the
chain of epiligrin
predicts two distinct polypeptides with identical COOH-terminal
domains, homologous to domain I+II and domain G of laminin
chains, and totally divergent amino-terminal domains. The isoform
3
substitutes a short arm, thus far uncharacterized,
for the truncated amino-terminal domain of the
3
counterpart (Ryan et al., 1994).
In this study, we
report the complete cDNA sequences of the 3 chains of murine
laminin 5, which demonstrate that the laminin
3B chain harbors a
short arm with unique structural features. We also provide evidence
that the laminin
3B and
3A isoforms display distinct
expression patterns.
Figure 1:
Schematic structure of overlapping cDNA
clones for 3A and
3B chains and domain structure of the
corresponding polypeptides. A, alignment of nine overlapping
cDNA clones and partial restriction map of the cDNAs. ATG indicates the
translation initiation signal and TAA the translation stop codon.
Restriction sites are EcoRI (R), HindIII (H), SmaI (S), and BamHI (B). The scale is shown in nucleotides. B, structure
of the
3A and
3B polypeptides with domain numbering in Romannumerals, according to Sasaki et
al.(1988). The EGF-like repeats composing the cysteine-rich
domains are represented by openboxes, numbered
according to Sasaki et al.(1988). The COOH-terminal domain G
is depicted by shadedboxes.
Figure 2:
A, deduced amino acid sequence of mouse
laminin 3B chain (upperline) aligned with the
partial sequence of human
3
(lowerline). Double horizontal traits between the compared
sequences underlined amino acid identities and single
horizontal traits indicate conservative substitutions. The arrow shows the putative signal peptide cleavage site. Cysteine residues
are boxed, and the potential N-linked glycosylation
sites (NX(S/T)) are indicated by fullcircles. Amino acid sequences with a putative biological
interest are underlined. Asterisk delimits the
carboxyl-terminal sequences common to laminin
3A and
3B
chains. Domains are boxed and labeled on the right. The sequence of the mouse
3B chain is available
from EMBL under accession number X84014. B, nucleotide
sequence of the cDNA encoding the amino-terminal domain specific to
mouse laminin
3A chain (upperline) and deduced
amino acid sequence (middleline). The amino acid
sequence of the human epiligrin
3
chain is also
reported (lowerline) (Ryan et al., 1994).
Conserved amino acid residues are indicated by a horizontalline and differing residues by the appropriate one-letter
code. Missing or mismatching amino acid residues are depicted by hatchedsquares. The putative cleavage site of the
peptide signal is indicated by a triangle. The mouse
3A
sequence is available from EMBL under accession number
X84013.
No significant homology is
found between regions of the large amino-terminal domain IV of the
3B chain and sequences in the amino-terminal domains of the
laminin isoforms characterized thus far. In particular, the conserved
sequences WWQS and Y(Y/F)YX
(G/R)G, located in the
amino-terminal domain VI of most of the laminin chains (Sasaki et
al., 1988; Hunter et al., 1989; Beck et al.,
1990; Kusche-Gullberg et al., 1992; Gerecke et al.,
1994; Vuolteenaho et al., 1994; Wewer et al., 1994),
are not found. Domain IV of laminin
3B chain has no significant
homology with domain IV of laminin
1 and
2 chains. However,
it displays 28% homology (42.6% if conservative changes are included)
with domain IV" (residues 872-1374) of Drosophila laminin
chain (Kusche-Gullberg et al., 1992). On the contrary,
the globular domain IVa of the
3B chain (residues 700-888)
displays 19.8% homology to domain IVa of mouse laminin
1 (residues
1143-1344) and is 12 amino acids shorter. Moreover, the EGF-like
domains IIIb of laminin
3B chain shows 47.2% homology with its
counterpart in laminin
1 chain and is 249 amino acids shorter (Table 1). The best alignment is obtained with a sequence
overlapping EGFs 7-11 of domain IIIb of laminin
1 chain
(between positions 981 and 1142) (Sasaki et al., 1988).
Domains IIIa of laminin chains
3B and
1 are 39.5% homologous.
In the
3 chain, domain IIIa retains the four EGFs that constitute
domain IIIa in the
1 chain (Sasaki et al., 1988).
However, EGF 4 comprises only 6 cysteine residues, which are found in
conserved positions (Sasaki et al., 1988). The size of the
different domains of the short arm of mouse laminin
3B chain and
their sequence homology with the corresponding domains of mouse and Drosophila
chains are summarized in Table 1.
The mouse 3B chain presents 77% homology to the available
sequences of the human
3B chain (Fig. 2A). 42
cysteine residues detected in the human
3B chain are conserved
between the two species; two extra cysteines (positions 1441 and 1585)
are found in the human sequence (Ryan et al., 1994). Domain I
+ II of the mouse laminin
3B chain (residues 1057-1647)
matches at 77% domain I + II of the human counterpart. In the
mouse, one residue (Lys-618) of the amino acid sequence is missing. A
protein adhesion Arg-Gly-Asp (RGD) sequence is found at position 1512
and matches the RGD sequence of the human polypeptide (position 658)
(Ryan et al., 1994). In the COOH-terminal globular domain G
(residues 1648-2568), subdomains G1 (residues 1648-1825),
G2 (residues 1826-1994), G3 (residues 1995-2209), G4
(residues 2210-2385), and G5 (residues 2386-2568) show 85,
73, 72, 69, and 73% sequence identity with the human
3B chain,
respectively. In mouse, subdomain G3 is one residue (Val-1341) shorter
than in man. Subdomain G1 (residues 1659-1661) contains a
putative motor neuron-selective adhesion site Leu-Arg-Glu (LRE) (Hunter et al., 1989), which is found in domain I-II of the human
3B chain (residues 369-371) (Ryan et al., 1994). In
human and mouse
3B chain, 11 glycosylation sites are in conserved
positions (Fig. 2A); however, the three glycosylation
sites found at positions 1209, 1326, and 1668 in the
3EpB chain
(Ryan et al., 1994) are substituted by the three glycosylation
sites at positions 912, 1398, and 1596 in the mouse sequence.
Figure 3:
Expression of laminin 3 chain
isoforms in lung and skin epithelial cells. 20 µg of
poly(A)
RNA from adult lung tissue (lane1) and epithelial cell line PAM212 (lane2) were successively hybridized with
P-labeled cDNAs probes M100, which codes for a peptide
common to both laminin
3A and
3B chains (panelA), M22, which is specific to the transcript
3B (panelB), and PR6H, which is specific to the
3A
transcript (panelC).
To verify this
possibility, we further investigated the expression of laminin 3A
and
3B isoforms at protein level. Western analysis was realized on
total extracts prepared from mouse skin and lung using the polyclonal
antibody SE152 specific to domain I+II of the two mouse
3
chain isoforms (Aberdam et al., 1994). In skin extracts, the
antibody detected a band with an apparent mass of 200 kDa and a
150-165-kDa band doublet (Fig. 4), which is the
electrophoretic migration pattern characteristic of the precursor and
mature forms of laminin
3A chain (Marinkovich et al.,
1992a; Aberdam et al., 1994). In lung extracts, antibody SE152
reacted with a single band with an apparent mass of 280-300 kDa (Fig. 4), which is a value concordant with the estimated
molecular weight of the polypeptide encoded by the full-length laminin
3B chain cDNA. It was thus clearly demonstrated that
immunoreactivity to the polyclonal antibody SE152 in mouse skin and
lung correlated with the presence in these tissues of mRNA for the
laminin
3A and
3B chains, respectively. These results are
therefore consistent with a cell type-specific expression of laminin
3A and
3B chain isoforms.
Figure 4: Western blot analysis of mouse skin (lane 1) and lung (lane 2) biopsies. 30 µg of protein extracts were fractionated on a 7.5% SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose filter, and reacted with polyclonal antibody SE152. The positions of molecular mass markers (kDa) are indicated. Exposure time was 2 min for lane1 and 30 min for lane2.
Figure 5:
In situ hybridization of mouse
fetal tissue sections with laminin 3A (A, C, and E) and
3B (B, D, and F)
antisense probes. Skin sections show the dermo-epidermal junction and
hair follicles with
3A probe (A) and negative staining
with
3B probe (B). Conversely, probe
3B stains
whisker pads (D), which are not labeled with
3A probe (C). Sagittal section of a 13.5-day embryo head revealing
negative staining with
3A probe (E) and strong staining
with
3B probe (F) is shown. Germinal layers of
telencephalon (t), choroid plexus (c), Rathke's
pouch (p), and mesencephalon (m) are shown. Bars: A and B, 35 µm; C and D, 50 µm; E and F, 500
µm.
In the present study, we relate the cloning of cDNAs coding
for the full-length 3A and
3B isoforms of mouse laminin
3 chain, and we demonstrate the tissue-specific distribution of
these laminin variants.
Sequence data reveal that the 3B chain
isoform (300 kDa) substitutes the amino-terminal short arm with two
alternating cysteine-rich domains and two globular domains for the
short amino-terminal peptide found in the
3A counterpart (200
kDa). These observations are concordant with previous results reporting
that human laminin
3A chain harbors a short arm, consisting of a
reduced thread-like structure comprised of four EGF-like repeats, and a
long arm, identical to the COOH-terminal regions of the
3B chain
isoform (Ryan et al., 1994). Apart from a restrained region
matching 29.7% of the amino acid sequence of domains III and IVa of
laminin
1 chain, the short arm of mouse laminin
3B chain
presents no homology with laminin
1 and
2 chains. The most
amino-terminal domain of the polypeptide displays a weak sequence
similarity with domain IV" of Drosophila laminin
chain,
which has been suggested to arise from the fusion of a duplicated
domain IV` of laminin
1 chain (Kusche-Gullberg et al.,
1992). However, in this, the two laminin
chains differ because no
homology is found between the
3B chain and laminin
isoforms.
The electron microscopy images of laminin 5 purified from
keratinocytes depict the molecule as a rod-like entity missing the
short arms characteristic of classical laminins or the globular
structures of the laminin 3B chain short arm. It has thus been
suggested that the
3B transcript corresponds to the
chain
polypeptide of laminin 6 (K-laminin) (Ryan et al., 1994).
However, lines of evidence suggest that the
chains of these two
laminins are distinct isoforms. First, the
chain of laminin 6 is
a truncated polypeptide lacking the amino-terminal short arm
(Marinkovich et al., 1992b). Second, the deduced molecular
mass (300 kDa) of polypeptide
3B is inconsistent with the
estimated mass of the
chain of laminin 6 (190 kDa) (Marinkovich et al., 1992b). No evidence for processing of the
chain
of laminin 6 that could account for this discrepancy has thus far been
provided. Third, synthesis of laminin 6 in H-JEB patients is not
affected by mutations in the LAMA3 gene, resulting in hampered
expression of
3A and
3B transcripts (Vidal et al.,
1995). Therefore, detection of the 300-kDa polypeptide corresponding to
the
3B transcript in lung epithelia, where the laminin
3A
chain is not detected, supports the assumption of the existence, and
the coexistence in some epithelia, of laminin 5 isoforms harboring
distinct
3 chains.
Thus far, information on laminin 5 has been
gathered from studies performed on the protein secreted by epidermal
keratinocytes. In the lamina lucida of the dermal-epidermal junction,
laminin 5 immunolocalizes with the anchoring filaments of
hemidesmosomes (Rousselle et al., 1991; Verrando et
al., 1993) and codistributes with integrin 6
4, the
transmembrane receptor associated with hemidesmosomes (Sonnenberg et al., 1991). The major importance of laminin 5 for the
formation of the hemidesmosomal adhesion complex and for the cohesion
of the dermal-epidermal junction is deduced from the observation that
in H-JEB an impaired expression of the protein correlates with
abnormality in hemidesmosome structures and extensive skin blistering
(Verrando et al., 1991). Herlitz JEB is also characterized by
disadhesion of gastrointestinal and lung epithelia in which
hemidesmosomal complexes have not been described (Jones et
al., 1994). Therefore, the possibility exists that, in gut and
basal membranes, laminin 5 incorporates the
3B chain to associate
with morphological structures distinct from hemidesmosomes via the
globular domains of the short arm of the
3B chain. This would be
consistent with the detection of laminin
3B chain in brain tissues
where laminin 5 may assume roles other than epithelial adhesion.
The
intense expression of the 3B transcript in neuroectoderm and
cerebellum confirms previous studies on expression of laminin 5 during
organogenesis, suggesting a role in brain and nerve development
(Aberdam et al., 1994). Laminin
1 chain harbors the
peptide SIKVAV that mediates cell attachment, migration, and neurite
outgrowth (Tashiro et al., 1989). Since in the mouse laminin
3 chain the peptide is not conserved, the sequence divergence
within this area may reflect a functional difference in the laminin
3 chain. The murine laminin
3A chain, however, is focally
expressed in the developing trigeminal nerve, which strengthens the
idea that this polypeptide may play a role in the migration and
polarization of motor neurons. The laminin
2 chain, which directs
the growing axons of intraspinal commissural neurons via the motor
neuron-selective adhesive site (LRE), is expressed in the central
nervous system (Sanes et al., 1990; Aberdam et al.,
1994). Interestingly, the human and murine
3 chains contain LRE
sites, which suggests that this isolaminin may be physiologically
active in the development of areas of the central nervous system.
However, the possible function of laminin 5, or that of laminin
isoforms comprising
3 chains, in the development of the central
nervous system deserves further investigations and accurate clinical
evaluation of JEB patients with mutations in the LAMA3 gene.
From
our results, it seems likely that structural variants of the 3
chain may contribute to regulate diverse functions of laminin 5.
Cloning of the cDNAs for the murine laminin
3 chains sets the
stage for experiments on gene disruption in mice embryonic stem cells
for the analysis of the specific role of the laminin
3A and
3B chain isoforms.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X84013 [GenBank]and X84014[GenBank].