* The Cutaneous Biology Research Center, Massachusetts General Hospital, and the Department of Dermatology, Harvard
Medical School, Charlestown, Massachusetts 02129; and The Departments of Neuroscience, Anatomy and Cell Biology, and
Ophthalmology, Tufts University School of Medicine, Boston, Massachusetts 02111
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Abstract |
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Laminins are heterotrimeric molecules composed of an , a
, and a
chain; they have broad functional roles in development and in stabilizing epithelial
structures. Here, we identified a novel laminin, composed of known
and
chains but containing a novel
chain,
3. We have cloned gene encoding this chain, LAMC3, which maps to chromosome 9 at q31-34. Protein and cDNA analyses demonstrate that
3 contains
all the expected domains of a
chain, including two
consensus glycosylation sites and a putative nidogen-binding site. This suggests that
3-containing laminins
are likely to exist in a stable matrix.
Studies of the tissue distribution of 3 chain show
that it is broadly expressed in: skin, heart, lung, and the
reproductive tracts. In skin,
3 protein is seen within
the basement membrane of the dermal-epidermal junction at points of nerve penetration. The
3 chain is also
a prominent element of the apical surface of ciliated
epithelial cells of: lung, oviduct, epididymis, ductus deferens, and seminiferous tubules. The distribution of
3-containing laminins on the apical surfaces of a variety of epithelial tissues is novel and suggests that they
are not found within ultrastructurally defined basement
membranes. It seems likely that these apical laminins
are important in the morphogenesis and structural stability of the ciliated processes of these cells.
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Introduction |
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LAMININS are large glycoproteins found in all basement
membranes (Ekblom, 1996). In overall appearance,
laminins are cross-shaped with a single long arm
arising from the coiled-coil interaction of three genetically
distinct polypeptide chains and three NH2-terminal short
arms, each originating from the individual polypeptide
chains (Maurer, 1996). The three subunit chains are
termed ,
, and
according to the current nomenclature
(Burgeson et al., 1994
). The complete primary structure
for each of the nine human laminin subunit chains has
been elucidated:
1 (Haaparanta et al., 1991
),
2, (Vuolteenaho et al., 1994
),
3 (Ryan et al., 1994
),
4 (Iivanainen et al., 1995a
),
1 (Pikkarainen et al., 1987
),
2 (Wewer et al., 1994
; Iivanainen et al., 1995b
),
3 (Gerecke et al., 1994
),
1 (Pikkarainen et al., 1988
), and
2 (Kallunki et al.,
1992
). The complete cDNA sequence for a fifth laminin
chain has been determined in mouse (Miner et al., 1995
);
partial cDNA sequences of human
5 (Durkin et al.,
1997
), and a novel chicken
chain (Ybot-Gonzalez et al.,
1995
) have been reported. All three chains have globular domains separated by multiple epidermal growth factor-like domains within the NH2-terminal short arms. Their
long arm portions are composed of heptad repeats that are
typical for
-helical coiled-coil proteins. In addition, the
COOH terminus of each
chain is composed of five globular (G)1 domains (Engel, 1992
).
The many functions ascribed to laminins are thought to
derive from their structural and signal transduction roles,
by which they contribute to the formation and stability of
basement membranes, to the stability of cellular attachments to basement membranes, and to cytoskeletal rearrangements mediated by their occupancy of cell surface
receptors (Ryan et al., 1994). These activities at least partially result from: (a) the binding of the COOH-terminal laminin G domains to integrins in most cells (Deutzmann
et al., 1990
; Drago et al., 1991
; Goodman, 1992
; Matter and
Laurie, 1994
; Rousselle et al., 1995
; Chen et al., 1997
), and/or
to dystroglycan in muscle cells (Henry and Campbell,
1996
; Pall et al., 1996
; Wewer and Engvall, 1996
; Cohen et al.,
1997
); (b) from the self-assembly of the laminins into a
pericellular extracellular matrix through interactions of
domains VI, which are present at the ends of the short
arms of the subunit chains (Yurchenco et al., 1992
; Yurchenco and Cheng, 1993
, 1994
); and (c) from assembly of
the pericellular laminin network with a conceptually separate network of type IV collagen molecules specifically
mediated by the molecule nidogen, one end of which binds
the laminin
1 chain, and the other end of which binds
type IV collagen and other basement membrane matrix components (Fox et al., 1991
; Battaglia et al., 1992
; Aumailley et al., 1993
; Reinhardt et al., 1993
).
The role of laminin 5 (3
3
2) in stabilization of epithelial-stromal interactions is the exception to this generalized scheme. While the 5 laminin G domains of
3 bind integrins
6
4 and
3
1 on the epithelial basolateral surface
(Niessen et al., 1994
), the absence of domains VI on the
truncated short arms of
3 and
2 (Kallunki et al., 1992
;
Ryan et al., 1994
), and the absence of a nidogen binding
site on
2 (Mayer et al., 1995
) prevent their participation
in the above-described model. Instead, the NH2 terminus of the epithelial cell-associated laminin 5 binds type VII
collagen present within the subjacent stromal matrix
(Rousselle et al., 1997
). Thus, laminin 5 appears to play a
unique role in epithelial frictional resistance, rather than a
direct role in overall basement membrane structure.
The primary functions of the laminin 1 chain in the
above generalized scheme is the contribution of a domain
VI which shares high amino acid sequence identity to domains VI of the laminin
and
chains and a unique sequence required for the binding of nidogen. This sequence, NIDPNAV, is present in the fourth EGF-like repeat of domain III (Mayer et al., 1979
; Poschl et al.,
1994
). In the
2 chain, the analogous sequence NVDPSAS
is present, but does not support high affinity nidogen binding (Mayer et al., 1995
).
In this report, we describe a novel human laminin chain. Its predicted structure indicates the presence of all
the domains homologous to
1, including a domain VI homologue and a nidogen-binding site containing a single
conservative amino acid substitution. We call this chain
the laminin
3 chain. The predicted structure of this chain
suggests that it should be capable of associations with
other laminin chains for basement membrane assembly;
however, immunolocalization studies in several tissues indicate the presence of laminin
3 chains in regions lacking
ultrastructurally identifiable basement membranes.
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Materials and Methods |
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Isolation of a Novel Laminin 12 (2
1
3)
The purification of the novel laminin 12 (2
1
3) was carried out as follows. Human placental chorionic villi were frozen in liquid nitrogen,
ground in a Waring blender, and then washed in 1 M NaCl. Unless otherwise noted, all subsequent steps were performed at 4°C. The final tissue
pellet (200 g, wet weight) was suspended by stirring for 48 h in 1 liter of
extraction buffer (0.5 M NaCl, 10 mM EDTA, and 625 mg/liter N-ethylmaleimide, 150 mg/liter phenylmethylsulfonyl fluoride, and 50 mM Tris-HCl, pH 7.8). The soluble fraction was collected after centrifugation (30,000 g, 60 min) and precipitated with 300 g/liter ammonium sulfate.
The precipitated proteins were collected by centrifugation (30,000 g, 60 min) and dissolved in chromatography buffer (2 M urea, 25 mM NaCl, 5 mM
EDTA, and 50 mM Tris-HCl, pH 7.8). The sample was then dialyzed
against the same buffer. After dialysis, 0.5 vol of buffer-equilibrated
DEAE-cellulose (DE-52; Whatman) was added and the mixture was
shaken overnight. Material not bound to DEAE-cellulose was collected
by filtration on a Buchner funnel (Whatman; filter 4) and precipitated by
addition of 300 g/liter ammonium sulfate. The proteins were collected by
centrifugation (30,000 g, 60 min), redissolved in Concanavalin A buffer
(0.5 M NaCl, 5 mM CaCl2, 5 mM MgCl2, and 50 mM Tris-HCl, pH 7.8),
and dialyzed against the same buffer overnight. The fraction was applied to a 2.5 × 5 cm Concanavalin A-Sepharose column (Pharmacia). Unbound material was removed by extensive washing while bound proteins were eluted by successive washing with 10 mM
-D-mannopyrannoside, 1 M
-D-glucopyrannoside, and finally 1 M
-D-mannopyrannoside (Sigma
Chemical Co.). Laminins are typically recovered in the latter two fractions; each fraction was independently concentrated to 10 ml with an Amicon concentrator (30-kD membrane) and applied to a 2.5 × 100 cm
Sephacryl S-500 column in 0.5 M NaCl, 50 mM Tris-HCl, pH 7.8. The fractions of interest were pooled, dialyzed against Mono-Q buffer (0.1 M
NaCl and 25 mM Tris-HCl, pH 7.8), and applied to a 1 × 5 cm Mono-Q
column (Pharmacia). Elution was achieved with a 60-ml 0.1-0.5-M NaCl
gradient. The fraction eluted at 250 mM NaCl was taken for further study.
Protein Sequencing
Protein sequencing was performed with minor modifications of published
methods (Aebersold et al., 1987). In brief, laminin 12 was resolved on a
polyacrylamide gel in the presence of 2-mercaptoethanol. The bands at
205, 185, and 170 kD were excised separately, digested with trypsin, and
then separated by HPLC and sequenced on an Applied Biosystem sequenator. Analysis of a trypsin digest of laminin
2 isolated from laminin
12 was performed with matrix-assisted laser desorption time-of-flight
mass spectrometry performed on a Finnigan Lasermat 2000 (Chait and
Kent, 1992
).
cDNA Cloning
By comparison of the laminin 1 amino acid sequence (SWISS-PROT accession number, P11047) with the dbEST database (Boguski et al., 1993
;
NCBI) using the program BLAST (Altschul et al., 1990
) one clone (these
data are available from GenBank/EMBL/DDBJ under accession number
AA297192) was chosen as a possible candidate for a new laminin
chain.
To extend the cDNA, specific primers for 5' or 3' extension were deduced
from a previously published expressed sequence tag (clone, AA297192).
Nested PCR on placental Marathon-Ready cDNA (Clontech) were
performed following the manufacturer's instructions using the supplied
nonspecific primers with the following gene-specific primers: for the
5' extension; in the first round, (5'-dCGCATGTGCCGTTCTCGTGGCACTGG); in the second round, (5'-dGCGGCAGGTGCACTGTCCAGTCTTGG); for the 3' extension, in the first round, (5'-dTGCACGGGACTGCAGCCGCTGCTACCC); in the second round
(5'-dGCTGCTACCCTGGCTTCTTCGACCTCC). For PCR, the Long
Expand PCR Kit (Boehringer Mannheim) was used with the following
conditions: denaturation, 94°C for 3 min; 10 cycles of 94°C for 30 s, 63°C
(
0.5°C per cycle) for 30 s, 68°C for 4 min; 25 cycles of 94°C for 30 s,
58°C for 30 s, 68°C for 4 min (+10 s per cycle); a final extension period at
68°C of 8 min.
The PCR samples from the first round were purified (PCR Purification
Kit; Qiagen) and 2% of the sample volume was used in the second round
of PCR using the same PCR protocol. These PCR products were purified
from an agarose gel (Gel Purification Kit; Qiagen) and either subcloned
(into PCR II or PCR 2.1 vectors; Invitrogen) or directly used for sequencing. To reconfirm the nucleotide sequence and control for PCR-induced
nucleotide substitutions, gene-specific primers were used to reamplify the
entire 3 cDNA. A first strand cDNA synthesis kit (Clontech) was used to
synthesize cDNA from total placental RNA using oligo dT, random, or
specific human laminin
3 antisense primers following the manufacturer's protocol; PCR was used to generate overlapping clones complementary to
the entire human laminin
3 chain. Sequencing of all the obtained PCR
products revealed the nucleotide sequence of laminin
3 from what we
eventually inferred as nt 297 to nt 5020. However, all further 5' extensions
failed to extend the sequence further toward the 5' end.
Genomic DNA
The sequence of the 5' end of the cDNA was determined from the human
genomic P1 clone DMPC-HFF#1-1461F2, which was obtained from a
PCR-based library screen performed at Genome Systems, Inc. The oligonucleotide primers that were provided to Genome Systems specifically
amplify exon 2 of the human 3 gene (sense primer, 5'-dCCCCGCAGGGGAAGGCGGGTCCTG; antisense primer, 5'-dGGCTTATGAGATCACGTATGTGAG). To obtain the sequence of the missing 5'
end, the genomic clone was sequenced (in 4% DMSO) with gene-specific
antisense primers. The sequence of the laminin
3 5' untranslated region
was confirmed by RT-PCR from placental RNA in 4% DMSO, (sense
primer, 5'-dCGCGCGGCGTCGGTGCCCTTGACC; antisense primer
5'-dGCTTGTAGATGGCAAAGCTCTCAGG).
Nucleotide Sequencing
Nucleotide sequences were determined with a Thermo Sequenase cycle
sequencing kit and 33P-ddNTP (Amersham Pharmacia) using either the
M13 forward or reverse primers or gene-specific primers synthesized in
our laboratory. A 1:1 ratio of inosine to guanosine was included in the sequencing mix. Sequence data were assembled and manipulated using
Genetyx-Max 8.0 and Genestream-1 at http://genome.eerie.fr/home.html
(Software Development Co., Ltd.). The signal peptide cleavage site was
predicted using http://genome.cbs.dtu.dk/services/SignalP/ (Nielsen et al.,
1997).
Northern Blot Analysis
A 956-bp PCR product (nt 1316-2271) was generated (Long Expand PCR
Kit; Boehringer Mannheim) from placental cDNA, purified (PCR purification kit; Qiagen), and labeled with [33P]dCTP (NEN) using the rediprime DNA labeling system (Amersham). Without further purification,
the probe was denatured in the same buffer containing 1/10 (vol/vol) human Cot-1 DNA (Boehringer Mannheim), and 1/10 (vol/vol) sheared
salmon testes DNA (GIBCO BRL) at 94°C for 5 min then chilled before
use. Northern blots (Clontech) were prehybridized in 50% formamide, 5×
SSPE, 1× Denhardt's, 1% SDS, 10% Dextran-sulfate, 0.1 mg/ml salmon
sperm DNA (GIBCO BRL) at 42°C for 2 h the probe was added and hybridized for 20 h. The blot was washed three times in 2× SSC, 1% SDS at
42°C and two times in 0.1× SSC, 1% SDS at 42°C. Blots were placed on a
BioMax MR film (Kodak) with a BioMax TranScreen-LE intensifying
screen (Kodak) for 20 h at 70°C.
Recombinant Expression of Domain I of the 3 Chain
A cDNA encoding the COOH terminus of human 3 was cloned into the
HisTrx and pPEP-T vectors (kindly provided by Richard Kammerer, Biozentrum, Basel, Switzerland; based on the pET system; Novagen). The
HisTrx vector has a histidine-tagged bacterial thioredoxin cDNA as a carrier in front of the cloning site; pPEP-T has a piece of the coiled-coil domain of mouse
1 in front of the cloning site. The
3 cDNA fragment used
was amplified by PCR from human placenta cDNA (see cDNA cloning)
using primers that include the EcoRI adapters: forward, 5'-GCGGATCCGAGGAAGCTGAGCGGGTGGGTGCTG-3'; reverse, 5'-GCGAATTCTTACTGCCAGCTGGCACAGTTCTCGGG-3'. The resultant plasmids were transformed into BL21(DE3) pLysS bacteria (Novagen) and
fusion proteins were isolated according to the pET System manual
(Novagen). A recombinant fragment containing only histidine-tagged thioredoxin was similarly expressed and purified.
Antibody Production
The 170-kD band (i.e., 3 chain) was excised from the reducing SDS-PAGE gel described above and injected into a rabbit for antibody production following standard procedures (Harlow and Lane, 1988
). The resulting serum (R16) was evaluated by Western analysis and shown to react
with the 170-kD
3 chain, and showed minor cross-reactivity with other
laminin chains at high antibody concentrations. All antibody-related studies presented in this communication were conducted at concentrations
well below those where cross-reactivity was observed. The histidine-tagged, thioredoxin-
3 fusion protein was used for the production of a
second rabbit antiserum (R21) which reacted with a single band in Western blots of placental extracts.
Affinity Purification
The R16 antiserum was affinity-purified by binding to gel-purified 3 that
had been transferred to nitrocellulose and then eluted with 1 M acetic acid
followed by immediate neutralization. The R21 serum was purified by
binding to the histidine-tagged, mouse
1-human
3 fusion protein coupled to activated CNBr-Sepharose; the bound antibodies were eluted with
2 M urea in PBS, or with 1 M acetic acid which was immediately neutralized. The immunofluorescent patterns produced by these two affinity-purified antibody pools were indistinguishable, and were similar to whole
R21 serum with reduced background staining. Only affinity-purified preparations of R21 serum were used for these studies. Antibodies made
against histidine-tagged thioredoxin were similarly isolated by affinity chromatography from R21 serum; immunofluorescent patterns with these
controls were blank.
Immunofluorescent Analyses
Most tissues were obtained from various colleagues using specimens for
other purposes: these include tissues from male and female rats; from normal human tissues discarded after surgery; and from rhesus monkey,
Macaca mulatta. Bovine tissues were purchased from a local slaughterhouse. Dissected and blocked tissues were placed directly in embedding
compound (O.C.T.; Sakura Finetek) and frozen by immersion in liquid nitrogen-cooled isopentane. 10-µm sections were made on a Leica CM 3000 or 3050 and collected on Superfrost slides (Fisher Scientific). Sections
were air dried and stored at 20°C until use. Just before use, sections
were immersed in acetone at
20°C and then rinsed three times in PBS at
room temperature. Sections were incubated with primary antibodies diluted in PBS containing: 2% normal goat serum, 0.25% sodium azide, and
0.1% Triton X-100. Sections were incubated overnight at 4°C; they were
washed in three changes of PBS (5 min per wash) and then incubated for
45-60 min with secondary antibody coupled to either Cy3, FITC, or Texas
red. After incubation, sections were washed and coverslipped in Prolong
(Molecular Probes). The sections were imaged on a Leica confocal laser
scanning microscope (Leica TCS-NT). The gain was adjusted in each
channel of the confocal to assure that there was no bleeding across the
channels; this adjustment is performed at the outset of each confocal session. Images were transferred to Adobe Photoshop and cropped for reproduction. The brightness and contrast were adjusted to make printed
images similar to that obtained on the microscope monitor.
Other primary laminin reactive primaries used were: polyclonal anti-EHS-laminin-1 (Sigma Chemical Co.); monoclonal anti-laminin 2 chain
(mAb 1922, Chemicon); polyclonal anti-laminin
4 (Miner et al., 1997
);
polyclonal anti-laminin
5 chain (Miner et al., 1995
); two monoclonal anti-laminin
1 chain (545, Marinkovich et al., 1992
; clone C21, Green et al.,
1992
); monoclonal anti-laminin
2 chain (Verrando et al., 1987
). Monoclonal anti-PGP 9.5 (Ultraclone, Ltd.) was used to identify nerves in skin.
Secondary antibodies used were: goat anti-rabbit FITC (ICN Pharmaceuticals); goat anti-rabbit-Cy3 (Jackson ImmunoResearch Laboratories).
In Situ Hybridizations
Paraffin sections were processed for in situ hybridizations as previously
described in detail (Libby et al., 1997). In brief, cRNA probes for the laminin
3 chain were generated from human
3 clones; cRNAs were labeled
during transcription by the incorporation of digoxigenin-UTP (Boehringer Mannheim); ~1 µg/ml of cRNA was used for hybridization; hybridizations were performed at high stringency (50% formamide and 5× SSC,
60°C; see Libby et al., 1997
for complete details). After overnight hybridization, sections were washed (50% formamide, 1× SSC, for 30 min at
60°C) and the unhybridized probe was destroyed by RNase A. The hybrids were detected with an anti-digoxigenin antibody coupled to alkaline phosphatase (Boehringer Mannheim). Sections were incubated overnight with anti-digoxigenin diluted 1:1,000 in blocking solution (Boehringer Mannheim). After washing to remove unbound antibody, endogenous alkaline phosphatase activity was blocked by washing in levamisole for 10 min; the alkaline phosphatase reaction was carried out overnight at room temperature.
Other Methods
SDS-PAGE (Laemmli, 1970) and electrophoretic transfer of proteins to
nitrocellulose with immunoblot analysis were performed essentially as
previously described (Lunstrum et al., 1986
). For the FISH analysis, a
1217 bp cDNA fragment was generated by RT-PCR from placental RNA,
using the sense primer 5'-dAGTGCCACTATAACGGCACATGCG and
antisense primer 5'-dCTCGTGTCTGCAAGGAGTCTGTCA. The gel
band was purified and subcloned (PCR II vector; Invitrogen). After the sequence of the fragment was verified, the resultant plasmid was used
for the fluorescent in situ localization of the LAMC3 gene (SeeDNA Biotech, Inc.).
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Results |
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Characterization of Laminin 12 (2
1
3)
Laminin 12 was extracted from human chorionic villi using
EDTA and partially purified by a combination of DEAE-cellulose, Concanavalin A, Sephacryl S-500, and Mono-Q
chromatography (see Materials and Methods). The final
fraction of interest resulting from the above protocol contains multiple laminins. Laminin 12 was resolved from this
mixture by SDS-PAGE (3-5% polyacrylamide) under
nonreducing conditions. Six bands were resolved (Fig. 1, Unreduced). Only the bands at ~560 kD and at the top of
the gel were reactive with a polyclonal anti-laminin antiserum (Sigma Chemical Co.; not shown). Therefore, the resolved band at 560 kD was excised, reduced in 10% 2-mercaptoethanol SDS-PAGE sample buffer, and resolved by
5% SDS-PAGE. Three bands were observed with masses
of ~205, 185, and 170 kD (Fig. 1, Reduced). The band at
185 kD reacted with a monoclonal antibody (clone 545;
Marinkovich et al., 1992) specific to the laminin
1 chain
(Fig. 1, Western blot). Each of the three bands was digested with trypsin, the peptides were resolved by HPLC,
and selected resolved peptides were sequenced. The sequences obtained are shown in Table I. The 205-kD chain
contained three peptides sequence identical to human
laminin
2 (published residues 536, 70, 1367; Vuolteenaho et al., 1994
). On that basis, the band was identified as human laminin
2, despite our observation that the 205-kD
band did not react with anti-
2 mAb (mAb 1922; Chemicon). The band at 185 kD produced two peptides identical
to human
1, and was thereby confirmed as human
1.
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In contrast to the easy identification of the other two
bands, the band at 170 kD contained three sequences not
contained within any known laminin chain. The NH2-terminal sequence of the 170-kD chain was determined, and
it also was novel; i.e., nonidentical to known laminin sequences. As these four sequences from the 170-kD band
were derived from an unknown laminin and we had identified the laminin and
chains, we assumed these sequences were derived from a novel laminin
chain that we
call
3.
The apparent molecular masses for the 205- and 185-kD
bands are not consistent with the literature values published for the 2 and
1 chains, respectively. Thus, these
bands are indicated in Fig. 1 as
2t,
1t, and
3t to indicate
that they have been processed (truncated). Laminin 2 and
laminin 4 were also present in these preparations; when
characterized by similar procedures (not described here in
detail) they showed molecular masses consistent with literature predictions, suggesting that our preparations were
not extensively and nonspecifically degraded. Together
these observations suggest that the truncations observed
for the
3-containing molecules may be physiologically relevant.
Characterization of the 3 cDNA
The cDNA sequences of human 1 and
2 were used to
probe the National Center for Biomedical Information
(NCBI) expressed-sequence-tag database (dbEST), and a
clone was identified that was homologous, but not identical, to
1 and
2. The sequence of this clone was used to
design PCR primers for extensions at 3' and 5' ends (see Materials and Methods) using human placental cDNA,
and additional sequence information was obtained by a
combination of genomic DNA and placental cDNA sequencing. The resulting sequence is shown in Fig. 2. The
deduced amino acid sequence contains regions with 100%
identity to all three of the peptide sequences obtained
from the 170-kD band (underlined in Fig. 2). The nucleotide sequence reported in this paper has been submitted
to GenBank/EMBL Data Bank with the accession number
AF041835.
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The DNA sequence contains an open reading frame
predicting 1620 amino acids, including a 19-amino acid-long putative signal peptide that closely meets the criteria
described by Nielsen et al. (1997). The predicted cleavage
site was confirmed by protein sequencing of the
3 NH2
terminus; this sequence exactly matched the predicted
amino acid sequence following the signal peptide. The
overall sequence of
3 is most similar to that of
1, sharing 52% amino acid similarity with human
1 (Pikkarainen et
al., 1988
). In addition, the amino acid sequence predicted
by the
3 cDNA contains a domain distribution most like
that of the
1 chain. All six domains are represented.
Overall, the 3 chain has 43.6% amino acid identity with
the
1 chain and 34% identity with the
2 chain. The highest conservation is seen between domains
1VI and
3VI
(Fig. 3). Domains
3V and III also show considerable similarity to domains
1V and III and
2V and III.
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The predicted 3 sequence contains nine potential glycosylation sites (Fig. 2, boxed), only two of which (Fig. 2,
boxed and underlined) are conserved in both human and
mouse
1. As these conserved sites are contained within
the globular domains IV and VI, it is likely that these sites
are used physiologically. There is a single RGD sequence
(boxed, hatched) within domain II, but this site is not conserved in either human or mouse
1 and
2 proteins. The
sequence NVDPNAV (Fig. 2, double boxed) occurs
within the fourth EGF-like repeat of domain III and is
a homologue of the nidogen binding site (NIDPNAV)
within the same domain of
1. These sequences differ by
only a single conservative amino acid substitution.
LAMC3 Maps to Chromosome 9q31-q34
The 3 chromosomal location was determined by searching the NCBI Human Genomic Sequencing Index data
base with the
3 cDNA sequence. The sequence is identical to a database, Sequence Tagged Sites (clone WI-14302),
that has been localized to chromosome 9q33-q34. A 1.2-kb
3 cDNA probe within domains I and II of the predicted
protein, the regions of least homology among the
chains, was used to localize LAMC3 by fluorescent in situ hybridization (FISH) analysis (SeeDNA Biotech, Inc.). The results confirm the localization to chromosome 9q31-q34
(Fig. 4).
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Laminin 3 Associates with
2
1 to Form Laminin 12 which, in Placenta, Is Lacking Part of the I/II Domains
of All Three Chains
To determine the domains present within 2t, the 205-kD
gel band, purified from placenta, was fragmented with
trypsin and the resulting peptides were fractionated by
HPLC; the masses of the eluted peptides were determined
by mass spectroscopy. The ion chromatograms were then
evaluated relative to the masses predicted from the published amino acid sequence for
2 in order to determine the NH2- and COOH-terminal peptides present within the
digest. The results identified a number of tryptic peptides;
among these, the peptide LVEHVPGQP(VR), beginning
at residue 70 within domain VI, was the most NH2-terminal; the peptide GTTMTPPADLIEK, beginning at
residue 1367 within domain III, was the most COOH-terminal. These results indicate that
2t is a fragment containing the short arm of the laminin
2 chain. This conclusion is consistent with the observation that the initial
peptide sequence identified from
2t was within the short
arm domains (above).
1t and
3t are also short arm fragments, as all the peptide sequences determined for both species are present
within the short arm domains. However, the masses of
2t,
1t, and
3t are greater than predicted for the short arms
alone. In addition,
2t,
1t, and
3t are not separable by
gel electrophoresis without the reduction of disulfide
bonds. Therefore, this truncated laminin 12 molecule is
very likely to contain portions of domain II of all three
chains, as the interchain disulfide bonds should lie between these domains. It is of interest to note that domain
II of
3 contains three cysteinyl residues whose bonding
partners are not readily identified and are not present in
domain II of other laminin chains. These three cysteinyl
residues are conserved in mouse
3 (Albus, A., and R.E.
Burgeson, unpublished observation). Whether these cysteinyl residues could form intrachain, interchain, or intermolecular disulfide bonds that in some way contribute to
the cleavage of
3 chain-containing laminins is unknown.
Tissue Distribution of 3 Expression by
Northern Analysis
Tissue RNA blots and Master Blot dot blots (Clontech)
were probed with a 3 nucleotide probe (nt 1316 to 2277).
A single major transcript of ~5 kb, consistent in size with
other laminin
chains, is present in several of the tissues
examined (Fig. 5 A). A small amount of a second larger
transcript can also be detected. This larger transcript is
most likely due to differences in polyadelylation or due to
inefficient splicing. The
3 chain RNA is abundant in spleen, testis, placenta, lung, and liver; lesser amounts are seen in kidney and ovary (Fig. 5 A). The predominance of
a single transcript allowed use of the RNA Master Blot
(Clontech) to determine expression in a large number of
other tissues. On this dot blot, tissue RNA concentrations
have been normalized to housekeeping genes. The Master
Blot (Fig. 5 B) confirms the abundant presence of
3 transcripts in placenta, adrenal gland, testis, lung, and fetal
kidney, but also shows detectable levels of
3 transcripts in
numerous additional tissues, including brain and skeletal
muscle.
|
Characterization of the Immunospecificity of
Anti-Laminin 3 (R16; R21)
A polyclonal antiserum, R16, was made in a rabbit to the
3 chain excised from a reduced SDS-PAGE gel similar to
that shown in Fig. 1. Another, R21, was made to recombinant
3 protein (see Methods). The R16 antiserum recognizes the
3 chain on immunoblots of placental extracts,
but at very high antibody concentrations, it shows some reactivity with the
1 and
1 chains as well. Thus, as a control, human neonatal foreskin was immunostained with
anti-laminin
1 (polyclonal anti-laminin 1; Sigma Chemical Co.), anti-laminin
2 (GB3, Verrando et al., 1987
), and
with anti-laminin
3 (R16). Crisp, brilliant fluorescence
was observed along the dermal-epidermal junction, and
around capillaries with the anti-
1 antibodies (data not
shown), and in the basement membrane at the dermal-epidermal junction with anti-
2 (data not shown); in contrast,
no signal above background was detected using the anti-
3
reagent (R16) when it was applied at dilutions of 1:250 or
more (data not shown). The antigen could not be unmasked by treatment of the cryosections with 2, 4, or 6 M
urea, or with 2 M guanidinium-HCl (data not shown). As
all known laminin chains have been detected in skin within
either the epithelial basement membranes or the vascular basement membranes, these results indicate that the cross-reactivity detected by Western blot analyses using the
polyclonal anti-
3 (R16) antibody was either not apparent
by immunohistochemistry, or was below detection at the
antibody concentrations used. For the subsequent anatomical experiments (below), R16 was diluted 1:250 or greater
to assure no cross-reactivity was occurring. R21, the affinity-purified antiserum to recombinant
3, was also tested
on sections of neonatal foreskin. As with R16, no immunoreactivity was seen (data not shown); thus we conclude
that this antiserum has no cross-reactivity with other
known
chains. Neither R21 nor R16 antiserums label the
blood vessel basement membranes (see below) consistent
with a lack of cross-reactivity to other
chains.
3-containing Laminins Are Localized to Peripheral
Nerves and to Ciliated Epithelial Apical Surfaces
Unlike the lack of anti-laminin 3 chain immunoreactivity
seen in neonatal foreskin, laminin
3 chain immunoreactivity was detected in human leg skin. As shown in Fig. 6
A, and consistent with published results, laminin
1 chain
reactivity is seen at the dermal-epidermal junction and
within the basement membranes of the vasculature, while
laminin
2 chain immunoreactivity is restricted to the dermal-epidermal junction (Fig. 6 B). The laminin
3 chain
immunoreactivity is further restricted to distinct patches widely spaced along the dermal-epidermal junction (Fig. 6
C). In experiments not shown, the immunoreactivity did
not correlate positively or negatively with sites of cell proliferation, nor did it correlate with fixed positions relative
to the rete ridges. However, there is a direct correlation of
the laminin
3 chain immunoreactivity (Fig. 6 D) with sites
where nerves cross the dermal-epidermal junction as detected by an antibody to the neuronal marker PGP9.5
(Fig. 6 E), which reacts with ubiquitin COOH-terminal hydrolase (Day et al., 1990
). The results in skin suggest that
3-containing laminins may be deposited into the dermal-
epidermal junction by nerve or nerve associated cells, or
that its expression by epithelial cells is induced by the adjacent nerve.
|
Laminin 3 is also expressed in the neural retina at the
apical surface of the retina and in the outer synaptic layer
(Libby, R.T., Y. Xu, E.P. Gibbons, M.-F. Champliaud, M. Koch, R.E. Burgeson, D.D. Hunter, and W.J. Brunken,
manuscript submitted for publication); in the retina, the
3
chain is coexpressed with the
4,
3, and
2 chains. Native
3-containing laminins have not been isolated as yet from
the retina; however, they have from another region of the
central nervous system, the cerebellum, from which we
have obtained two novel laminins,
3
2
3 and
4
2
3
(Champliaud, M.-F., unpublished observations). In addition, anatomical methods (immunohistochemistry and in
situ hybridization), demonstrate the expression of
3 in cerebellum and forebrain structures (Brunken, W.J., unpublished observations). It seems likely that
3-containing
laminins will be a general feature of the matrix in the CNS.
The Northern analysis indicated that the laminin 3
chain was most strongly expressed in placenta, testis, lung,
liver, spleen, and ovary. Therefore, we examined the localization of
3 chains within testis, lung, and ovary. The reactivity within the epididymis and the fallopian tube were
particularly striking. Thus, the distribution of
3 in these
tissues was extensively studied. In the female reproductive
system, the oviduct was strongly reactive. Cryosections of
the bovine (Fig. 7, A-F) or rat (Fig. 7, G-I) ampulla reacted for
3 using R16 (Fig. 7, A, D, and G-I), or R21
(Fig. 7, B and E) showed brilliant immunoreactivity at the
apical surfaces of the tubal mucosa. Double immunofluorescent studies performed with laminin
3 and either laminin
2 (Fig. 7, A and D) or laminin
5 antibodies (Fig. 7
E) demonstrated that both of these
chains are restricted
to the basement membranes of the tubal epithelial and the
subjacent endothelium whereas
3 is expressed at the apical surface. The pre-immune serum from rabbit 21 (Fig. 7
C) was negative, as was the reactivity of anti-thioredoxin
antibodies purified from the R21 serum by immunoaffinity
(Fig. 7 F).
|
The pattern of immunoreactivity for R16 in the rat oviduct was identical to that seen in bovine tissue (Fig. 7 G).
Higher magnification micrographs of the epithelial apical
surface of the rat ampulla (Fig. 7, H and I) show the 3
chain to be localized to the apical surface of the epithelial
cells at the base of the cilia.
It should be noted that the labeling pattern of R21 differed somewhat from that of R16. In general, the pattern
with R21 was somewhat punctate, showing large deposits
of immunoreactivity at the apical surface, and increased
cytoplasmic labeling of the tubal epithelium, whereas the
R16 immunoreactivity was more restricted to the apical
extracellular surface. These observations suggest that the
R21 antiserum, made to recombinant domain I, may recognize the unfolded 3 chain better than the R16 antiserum, which should recognize primarily short arm domains.
The male monkey reproductive tract was examined also.
Like the fallopian tube, the epithelium in the epididymis is
a single columnar epithelium (Fig. 8, A, H, and E). In situ
hybridization performed on adjacent sections of the monkey epididymis (Fig. 8 B; 3) localized transcripts for the
3 chain to the apical region of the epithelial cells (compare Fig. 8, A with B). R16 (data not shown) and R21 (Fig.
8, C and D, R21) sera gave similar patterns, reacting with
both the basal and apical surfaces of the epithelial cells.
The R21 antiserum reacted with apparently intracellular
stores of
3, as was seen in the bovine fallopian tube. The
preimmune control serum from R21 showed only punctate autofluorescence (Fig. 8 E, Pre).
|
Potential chains partners were explored by examination
of the same tissue with antibodies specific for a variety of
other laminin chains: 2 (Fig. 8 F),
4 (G),
1 (H), and
2
(I). We used two monoclonal antibodies to test for the
presence of
1 at the apical surface (clones 545; and C21)
both gave the same pattern of immunolabeling; only the
results with clone 545 are shown. As can be seen readily,
2 and
2 were restricted to the basal surface of the epithelial cells, while staining for
4 and
1 were also seen at
the apical surface. Thus, in contrast to the results from placental extracts,
4 (and not
2) appears to be a candidate
chain partner for
3 in the epididymis. These observations suggest that a wide variety of
3-containing laminins will
be expressed in a tissue-specific pattern.
Expression of laminin 3 chain was examined in the rat
as well and the tissue distribution of
3 in the rat epididymis was similar to that described for the monkey (data not
shown); namely,
3 immunoreactivity was localized to the
apical surface of the epithelium. We also studied other
regions of the rat reproductive system. Unlike laminin-1
immunoreactivity, which is localized to the basement
membrane of the seminiferous tubules (Fig. 9 A),
3 immunoreactivity is not present within the basement membrane of the seminiferous tubules nor is it found around
the interstitial cells (Fig. 9 A, arrows; Fig. 9 B, asterisk).
Within the seminiferous tubules, only the occasional tubule reacted strongly with the laminin
3 reactive serum
(R16, Fig. 9 B); it was our impression that those tubules
identified by the antibody contained nearly mature spermatids. Further along the male reproductive system, in the
ductus deferens, laminin-1 immunoreactivity (Fig. 9 C; arrows mark the apical surface of the epithelium) was seen
along the epithelial basement membrane, in the lamina
propria and ensheathing the smooth muscle cells of the
muscular layer. In contrast,
3 immunoreactivity (R16)
was found at the apical and basal surfaces of the epithelial
cells, as well as intracellularly (Fig. 9 D).
|
The apical distribution of the 3 chain is not confined to
the reproductive system; in rat lung, the ciliated epithelial
cells lining the bronchi were also strongly reactive with the
anti-laminin
3 antiserum, R16 (Fig. 9 E). Again, the fluorescence was apparent along the apical surface, as determined by differential interference contrast microscopy
(Fig. 9 F). No
3-immunoreactivity was seen in respiratory
epithelium nor in the pulmonary capillary bed (not shown).
![]() |
Discussion |
---|
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---|
The laminin 3 chain described here is the eleventh laminin subunit to be identified. The predicted primary and
secondary structure of this chain suggests that
3 is more
closely related to human
1 than
2. Unlike
2, the
3
cDNA sequence predicts a laminin subunit without the
short-arm truncations predicted for
2. Perhaps more significantly,
3 contains a
1-like nidogen binding motif with
only a single conservative amino acid substitution, suggesting that
3-containing laminins should be capable of associating with other basement membrane molecules through
nidogen interactions (Mayer et al., 1995
; Poschl et al.,
1996
). In addition, domain VI of
3 shares the highest sequence identity with domain VI of the
1 chain. As this
latter domain has been shown to support laminin self-assembly (Yurchenco and Cheng, 1994
), it seems reasonable to suggest that domain VI of the
3 chain may also
support self-assembly.
Two of the predicted glycosylation sites in the 3 chain
are also found in human and in mouse
1; these are within
short-arm globular domains, i.e., VI and IV. Interestingly,
the glycosylation site in domain IV is also found in human
(Kallunki et al., 1992
) and in mouse (Sugiyama et al.,
1995
)
2. This remarkable conservation of glycosylation
sites among these three chains and between these species
suggests that these sites are indeed important and glycosylated; they are likely to be critical in the folding of this region or may play another important function.
The RGD sequence within domain II of the 3 chain is
found in neither
1 nor in
2. Moreover, it seems likely
that this sequence is not functional within native
3-containing laminins as it is located within the coiled-coil
region of
3. However, it very well may promote integrin-mediated recognition of non-native molecules or of protein fragments.
In placenta, the 3 chain can combine with the laminin
2 and
1 chains. This observation suggests that, unlike
2
which pairs preferentially with
3,
3 may pair with any
chain, with the possible exception of
3, and with any of
the known
chains. This prediction suggests the existence
of an additional 10 laminins with the following chain compositions:
1
1
3,
1
2
3,
2
1
3,
2
2
3,
3
1
3,
3
2
3,
4
1
3,
4
2
3,
5
1
3, and
5
2
3. In both
the epididymis and the fallopian tube,
3 is not combined with
2. In the epididymis, the
4 and
1 chains appear to
be potential partners. Given that the total number of human laminins is not known, at least one additional
chain
has been identified in chicken (Ybot-Gonzalez et al., 1995
;
Liu et al., 1998
) and in mammals (Olson, P.F., unpublished
observations), assigning a final laminin numerical identifier to these laminins is premature. However, as we have
shown
2
1
3 to be the twelfth laminin to be identified,
we provisionally call this heterotrimer, laminin 12.
The masses of the chains of laminin 12 as approximated
by electrophoretic mobility are considerably less than predicted by the amino acid sequences and from prior experience with the 2 and
1 chains. They are also less than the
2,
1, and
2 chains present in laminins 2 and 4 obtained
from the same preparations. The reason for these more
rapid electrophoretic migration rates appears to be proteolysis within the domains II of the chains comprising this
molecule. In placenta, this proteolysis may be physiological, since laminins 2 and 4 isolated from the same preparations are apparently intact. The significance of this observation awaits considerable additional experimentation
before it is understood. However, we have observed three
cysteinyl residues within domain II of the
3 chain that are
not present in other human
,
, or
chains. It is possible
that a disulfide bond between two of these residues distorts the coiled-coil conformation making molecules containing this chain more susceptible to proteolysis. At this time, we do not know if truncation of laminins containing
the
3 chain can be generalized to tissues other than placenta; however, this seems unlikely in that our isolation
of
3-containing laminins from the CNS do not show
the same truncation (Champliaud, unpublished observations). The COOH-terminal truncation of
2t explains its lack of reactivity with an anti-
2 antibody, mAb 1922, which is specific for the
2 G domain (Engvall et al., 1990
).
The antiserum to the recombinant 3 domain I fusion
protein (R21) was originally made to evaluate this potential processing, since epitopes contained within this domain should be absent from the processed molecule. In
this regard, the immunohistochemical data is not definitive. Like R16, R21 immunoreactivity is seen at the apical
surface; however, the apical reactivity is distinctly different than that for R16. Specifically, R21 immunoreactivity appears as a plaque-like structure at the cell surface, with
some reactivity within the cells. Thus, it seems possible
that R21 epitopes are entirely intracellular but it is also
possible that some of the R21 epitopes are present at the
apical surface of these epithelial cells. Further experimentation beyond the scope of this report is required to address this question. However, laminins containing
3
chains have been immunoisolated from the medium of
A204 cells derived from a human rhabdosarcoma (Champliaud, M.-F., unpublished data), indicating that
3-containing laminins are capable of being secreted in vitro.
These
3-containing laminins from A204 are a mixture
of processed (truncated) and unprocessed (untruncated) molecules.
Reports of laminins in tissue locations not identified as
basement membranes are increasingly frequent. In the
brain, laminins have been observed not only within the
basement membrane of capillaries, but also at other sites
not conceptualized as basement membranes (Higuchi et al.,
1991; Jucker et al., 1992
, 1996a
,b; Mori et al., 1992
; Tian
et al., 1996
, 1997
; Hagg et al., 1997
; Yamamoto et al.,
1997
). In the eye, the laminin
2 chain has been identified in both basement membrane and non-basement membrane locations (Hunter et al., 1992; Libby et al., 1996
;
Libby et al., 1997
; Toti et al., 1997
). Laminins have also
been observed in cartilage (Durr et al., 1996
).
Intriguingly, the laminin 3 chain appears most commonly to be associated with non-basement membrane
structures. In the cerebellum,
3 chains are detected in
the pericellular nets surrounding both neurons and glia
(Brunken, W.J., unpublished observations). Reported elsewhere (Libby et al., manuscript submitted for publication, see above),
3 is present within the neural retina at two extracellular sites: between the outer segments of the photoreceptors, and at the synapses of the photoreceptors with
the bipolar and horizontal cells. In the retina, these
3-containing molecules are the products of the Müller glial
cells which, like the tubal epithelium, contain a considerable store of intracellular
3 chain. The laminin
3 and
2
chains are also present at these sites, whereas the
1 and
2 chains are absent. The functions fulfilled by these laminins are unclear, but possibilities include: stabilization of
neural architecture; induction and stabilization of differentiated neural phenotypes (Hunter et al., 1992b
; Libby et
al., 1996
; Hunter and Brunken, 1997
); and stabilization of
synaptic junctions.
However, the most abundant expression of 3 as detected by Northern analyses is not within neural tissues,
but rather is in the testis, the placenta, the spleen, the lung,
and the ovary.
3 immunoreactivity is present at the bases
of the epithelial cilia of the epididymis, the trachea, the
bronchi, and the oviduct. There are no structures resembling basement membrane at these sites. However,
3
chains may be present within the basement membranes along the basolateral surfaces of some of these epithelia.
The chain partners for the
3 chain in these apical laminins are not yet known with certainty. However, in epididymis, the laminin
2 chain does not colocalize with
3
at the apical epithelial surface; rather, the
4 and
1
chains are present at that location and, thereby, are potential chain partners. Thus, it seems likely that
3 will be as
promiscuous as
1 with respect to partner choice during
laminin assembly.
The presence of laminins along ciliated epithelial surfaces was unexpected and their functions there are unknown. Perhaps a modified basement membrane containing at least laminin helps organize or stabilize the
specialized cytoskeleton of the cilia. Laminins at these apical surfaces may also participate in the anchorage of mucins to the surface. Alternatively, laminins might stabilize
the outfoldings of the plasma membranes of the cilia. Similar functions for laminins have been postulated to stabilize the junctional folds beneath synapses at neuromuscular junctions (Noakes et al., 1995), and contribute to the
organization of epithelial hemidesmosomes (Langhofer et
al., 1993
; Baker et al., 1996
). Laminins expressed at the
apical surface of the retina are thought to play a role in
photoreceptor morphogenesis, specifically outer segment formation and synapse development (Libby et al., 1997
,
1998
).
Consistent with the above speculations regarding an essential function for 3 containing laminins in neural tissues, the chromosomal locus of LAMC3 at 9q31-q34 is
shared with four diseases having various degrees of neural
dysfunction in common: Fukuyama congenital muscular
dystrophy (FCMD); muscle-eye-brain syndrome; Walker-Warburg syndrome; and retinitis pigmentosa-21 with deafness (RP-21). The genetic cause of the latter three of these
conditions is unclear. While
3 expression may be affected
in FCMD, the LAMC3 cannot be the genetic cause of the
problem as a retrotransposal insertion in a different gene
has recently been identified in 87% of the FCMD alleles
(Kobayashi et al., 1998
). However, LAMC3 is an excellent candidate for mutations underlying one or more of remaining syndromes in this cluster of human diseases, particularly RP-21.
We have identified the laminin 3 chain together with
the
2 and
1 chains within laminin 12 from human placenta, but multiple other combinations are possible in
other tissues. It would be of particular interest if
3 were
to associate with the
2 chain and well as with the
2
chain, as both these chains have been reported to show
neural and muscle-associated expression and function. The
3-containing laminins are likely to be the subject of
considerable interest as they constitute a novel class of
laminin molecules distributed outside of the traditional
basement membrane. The identification of the function of
this diverse family of laminins remains to be elucidated by
future experiments.
![]() |
Footnotes |
---|
Address correspondence to Dr. Marie-France Champliaud, MGH-East CBRC, Bldg. 149, 13th Street, Charlestown, MA 02129. Tel.: (617) 724-8285. Fax: (617) 726-4453. E-mail: marie-france.champliaud{at}cbrc2.mgh.harvard.edu
Received for publication 9 February 1999 and in revised form 23 March 1999.
The authors gratefully acknowledge the excellent technical support provided by Ms. Carol Milbury, and the expert assistance of Dr. Yimin Ge
with confocal microscopy. The authors also thank Dr. Richard Kammerer
for the HisTrx and a pPEP-T vector.
Dr. Brunken is on leave from the Department of Biology, Boston College.
This work was supported by the U.S. Public Health Service grants AR35689 (R.E. Burgeson), EY12037 (D.D. Hunter); the E. Matilda Ziegler Foundation (W.J. Brunken); and additional support from the Cutaneous Biology Research Center, Massachusetts General Hospital.
![]() |
Abbreviations used in this paper |
---|
FCMD, Fukuyama congenital muscular dystrophy; FISH, fluorescent in situ hybridization; G, globular.
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