©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Complete Primary Structure of the Mouse Laminin 3 Chain
DISTINCT EXPRESSION PATTERN OF THE LAMININ alpha3A AND alpha3B CHAIN ISOFORMS (*)

(Received for publication, May 8, 1995; and in revised form, July 12, 1995)

Marie-Florence Galliano (1)(§) Daniel Aberdam (1)(§)(¶) Adriano Aguzzi (2) Jean-Paul Ortonne (1) (3) Guerrino Meneguzzi (1)(**)

From the  (1)U385 INSERM, Faculté de Médecine, 06107 Nice Cedex 2, (2)Institüt für Neuropathologie, Universität Zürich, 8093 Zürich, Switzerland, and (3)Service de Dermatologie, Hôpital Pasteur, 06002 Nice Cedex 1, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have isolated and characterized overlapping cDNA clones encoding the alpha3A and alpha3B chains of mouse laminin 5. Sequence analysis of the cDNA for the alpha3B 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 alpha chains identified thus far. The short arm of the alpha3B 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 alpha3A 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 alpha3B isoform and displays 77% conservative homology to the alpha3Ep 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 alpha3A and alpha3B isoforms, and in situ hybridization on mouse embryos revealed a focal localization of alpha3B in areas of the central nervous system.


INTRODUCTION

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 alpha-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 alpha, beta, 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 alpha 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 (alpha3), 140 kDa (beta3), and 105 kDa (2). This mature species derives from a cell-associated molecule as a result of two extracellular processing events that generate the alpha3 and the 2 chains from distinct 200- and 155-kDa precursor polypeptides, respectively (Marinkovich et al., 1992a). The laminin alpha3 chain is immunologically related to a distinct laminin 190-kDa alpha chain synthesized by keratinocytes that interacts with a beta1 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 alpha6beta4 and alpha3beta1 (Carter et al., 1990; Marinkovich et al., 1992b). In amnios and fetal skin, the 190-kDa laminin alpha chain associates also with a beta2 and a 1 chain to form laminin 7 (Wewer et al., 1994).

Mutations in the genes encoding laminin 5, including its alpha3 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 alpha3 chain of laminin 5 identified two species of mRNA transcripts.^1 Partial sequence analysis predicts two polypeptides identical to the alpha3 and alpha3 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 alpha3beta1 in focal adhesion and with integrin alpha6beta4 in hemidesmosome adhesion structures (Carter et al., 1991). The cDNA encoding the alpha chain of epiligrin predicts two distinct polypeptides with identical COOH-terminal domains, homologous to domain I+II and domain G of laminin alpha chains, and totally divergent amino-terminal domains. The isoform alpha3 substitutes a short arm, thus far uncharacterized, for the truncated amino-terminal domain of the alpha3 counterpart (Ryan et al., 1994).

In this study, we report the complete cDNA sequences of the alpha3 chains of murine laminin 5, which demonstrate that the laminin alpha3B chain harbors a short arm with unique structural features. We also provide evidence that the laminin alpha3B and alpha3A isoforms display distinct expression patterns.


MATERIALS AND METHODS

Isolation and Analysis of cDNAs

A mouse lung cDNA library (Zap, Stratagene) was screened with a random-primed P-labeled (prime-it, Stratagene) cDNA clone MN97 that encodes a region of the I+II domain of the mouse laminin alpha3 chain (Aberdam et al., 1994). 12 positive phage clones were excised into pBluescript SK (Stratagene). The largest cDNAs, M100 (2.2 kb), (^1)MR9 (1.5 kb), and M2C3 (3.3 kb), were completely sequenced on both strands using a Sequenase kit (Pharmacia Biotech Inc.). To obtain additional clones, the cDNA library was rescreened at low stringency using the human cDNA NA12, which codes for the 3`-end of the human laminin alpha3 chain, (Vidal et al., 1995) as a radioactive probe. A positive clone MZ6b (2.5 kb) containing the 3`-end of the complete nucleotide sequence of the cDNA was thus identified. cDNA clones representing extensions of cDNA M2C3 were isolated by PCR amplification of the mouse cDNA expression library. The primers used for 5`-extension were as follows: 5`-CAGTAGCAACACACTCCTTA-3` (left) and 5`-CCAGGAGCACACTTGTC-3` (right), which correspond to a sequence in the vector T7 promoter and to a 5`-end sequence of cDNA MR9, respectively. The primers used for 3`-extension were as follows: 5`-CAGTAGCAACACACTCCTTA-3` (left) and 5`-TAGCCTGTGCCTTCAAAGTA-3` (right), which correspond to a 3`-end sequence of cDNA M2C3 and to a 5`-end sequence of cDNA MZ6b, respectively. After purification and subcloning of the amplification products into PCRTM-II vector (TA cloning kit, InVitrogen), clones M22 (960 nucleotides) and M0.2 (254 nucleotides) were completely sequenced. Clones representing the 5`-end of the complete nucleotide sequence of the laminin alpha3A and alpha3B cDNAs were obtained by 5`-rapid amplification of cDNA ends (5`-RACE kit; Life Technologies, Inc.). Briefly, 200 ng of poly(A)-selected RNA from the epithelial cell line PAM212 (Yuspa et al., 1980) was reverse transcripted using the primer 5`-TCGCAGTCATCACATTCTT-3`. The first strand DNA was amplified by PCR using a 5`-RACE kit as devised by the manufacturer with the provided sense primer (Anchor Primer) and the specific antisense primer 5`-CTGTGTTCCTGTGTATCCGG-3`, which corresponds to a sequence of cDNA M2C3. The 491-base pair PCR product was subcloned into the TA cloning PCRTM-II vector (InVitrogen) resulting in clone PR6, which contains the complete 5`-end of the laminin alpha3A isoform. Following a similar procedure, poly(A) RNA from mouse lung was retrotranscribed into cDNA using primer 5`-GACAGAAGGAGGCAAGGAAGGAACC-3`. PCR amplification was made using the Anchor Primer of the 5`-RACE kit and a specific antisense primer 5`-TTCACAATCACCTCAGT-3`, which corresponds to a 5`-end sequence of cDNA M22. The resulting MR10 clone (1145 base pairs) contains the complete 5`-sequence of the cDNA for the laminin alpha3B isoform. All of these clones were further sequenced on both strands. Primer extension analysis using the 5`-most fragment of clone MR10 and poly(A) RNA prepared from mouse lung tissue showed that the 5`-untranslated sequence did not extend beyond the 5`-region of this cDNA.

Cell Culture

PAM212, a mouse transformed epidermal cell line, was cultured in Eagle's minimal essential medium supplemented with 10% fetal calf serum.

Northern Blot Analysis

Total RNA was isolated from adult mouse lung tissues and from cultures of actively growing PAM212 cells (Chomczynski and Sacchi, 1987). 20 µg of poly(A)-enriched RNA was electrophoresed on 1.0% denaturing agarose gels, transferred onto nylon membranes (Hybond-N, Amersham), and hybridized with radioactive probes obtained by P random priming of the cDNA fragment PR6H, which corresponds to the 230-base pair HindIII restriction product of cDNA PR6, the full-length cDNAs M100 and M22 inserts.

Western Blot Analysis

Protein extracts of adult nude mouse skin and lung tissues were prepared as previously detailed (Vailly et al., 1994). Human normal keratinocytes and lung carcinoma cells were scraped at subconfluency and homogenized in 1% Triton X-100, phosphate buffer (0.1 M, pH 7.2) containing 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 50 mM NaF, and 1 mM EDTA. Protein content was determined by Bradford assay (Bio-Rad). Samples (30 µg of protein) were boiled in sample buffer (60 mM Tris, pH 6.8, 1% SDS, 10% glycerol, and 5% beta-mercaptoethanol) and separated by SDS-polyacrylamide gel electrophoresis (7.5% acrylamide gels). Following electroblotting, the nitrocellulose sheets were incubated in a blocking buffer (0.5% gelatine, 3% bovine serum albumin, 0.1% Tween 20, 1 mM EDTA in 10 mM Tris, pH 7.4, 0.15 M NaCl) to avoid nonspecific binding. The blots were then incubated with rabbit antisera directed against either mouse alpha3 chain (SE152) or human alpha3 chain (SE85) (Baudoin et al., 1994) and followed by incubation with peroxidase-conjugated second antibody. The antibody-antigen complex was revealed using the enhanced chemiluminescence (ECL) Western blotting kit (Amersham).

In Situ Hybridization Analysis

Sense and antisense probes specific to laminin transcripts alpha3A and alpha3B were obtained by labeling cDNAs PR6H, M22, and MZ6b with digoxigenin-uridine triphosphate (Boehringer Mannheim, France). In situ hybridization on mouse fetal tissues was performed using a method devised to detect mRNA transcripts in neural tissue (Schaeren-Wiemers and Gerfin-Moser, 1993).


RESULTS

Identification of cDNA Clones Encoding Mouse Laminin alpha3 Chain

Screening of a mouse lung cDNA library with a radioactive cDNA coding for the alpha3 chain of human laminin 5 identified cDNA clones corresponding to the full-length mouse laminin alpha3 chains (see ``Materials and Methods'' and Fig. 1A). Sequence analysis revealed that these cDNA clones represent two distinct transcripts, designated as alpha3A and alpha3B, homologous to the epiligrin alpha3 and alpha3 chain isoforms, respectively (Ryan et al., 1994). The schematic structure of overlapping clones is depicted in Fig. 1A.


Figure 1: Schematic structure of overlapping cDNA clones for alpha3A and alpha3B 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 alpha3A and alpha3B 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.



Nucleotide and Amino Acid Sequences of Laminin Isoform alpha3B

The complete amino acid sequence of the cDNA for the mouse laminin alpha3B chain is shown in Fig. 2A. The cDNA sequence contains an open reading frame (ORF) of 7704 nucleotides flanked by 5 nucleotides of 5`-untranslated sequence and 368 nucleotides of 3`-untranslated sequence. The 3`-non-coding sequence contains a polyadenylation signal (AAUAAA) located 16 nucleotides upstream of the poly(A) tail. The ORF encodes a protein of 2568 amino acids. The first in-frame ATG is in favorable context for initiation of translation (Kozak, 1991) and precedes a stretch of 26 hydrophobic amino acids typical of a signal peptide. According to the rule of Von Heijne(1986), a cleavage site was predicted following Gln-27. After cleavage of the signal peptide, the protein consists of 2541 residues with a predicted molecular mass of 279,510 Da. The mature protein has 11 putative N-linked glycosylation sites (Asn-X-Ser/Thr), and the molecular mass of the glycosylated peptide is estimated to exceed 300,000 Da.


Figure 2: A, deduced amino acid sequence of mouse laminin alpha3B chain (upperline) aligned with the partial sequence of human alpha3 (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 alpha3A and alpha3B chains. Domains are boxed and labeled on the right. The sequence of the mouse alpha3B chain is available from EMBL under accession number X84014. B, nucleotide sequence of the cDNA encoding the amino-terminal domain specific to mouse laminin alpha3A chain (upperline) and deduced amino acid sequence (middleline). The amino acid sequence of the human epiligrin alpha3 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 alpha3A sequence is available from EMBL under accession number X84013.



Domain Structure of Laminin alpha3B Chain

The laminin alpha3B chain is comprised of a short arm of 1056 amino acids, a long arm comprising a rod-like region of 589 residues and a carboxyl-terminal globular domain G of 920 residues (Fig. 2A). Recently, Ryan et al.(1994) reported the partial cDNA sequence for the alpha3 of epiligrin, which resulted identical to the sequence of the alpha3B chain of human laminin 5. (Vidal et al., 1995). Since the cDNA sequence for the amino-terminal region of human laminin alpha3B chain is not available, we compared the domain structure of the short arms of mouse laminin chains alpha3B and alpha1. Alignment with the amino acid sequence of mouse laminin alpha1 chain reveals that the polypeptide chain alpha3B harbors a truncated amino-terminal end missing the most amino-terminal domains V and VI. Therefore, the short arm of the chain comprises the cysteine-rich EGF-like domains IIIa (residues 889-1057) and IIIb (residues 498-699), which are predicted to have rigid rod-like structures, and domains IVa (residues 700-888) and IV (residues 27-497), which are predicted to form globular structures (Fig. 1B).

No significant homology is found between regions of the large amino-terminal domain IV of the alpha3B 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(7)(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 alpha3B chain has no significant homology with domain IV of laminin alpha1 and alpha2 chains. However, it displays 28% homology (42.6% if conservative changes are included) with domain IV" (residues 872-1374) of Drosophila laminin alpha chain (Kusche-Gullberg et al., 1992). On the contrary, the globular domain IVa of the alpha3B chain (residues 700-888) displays 19.8% homology to domain IVa of mouse laminin alpha1 (residues 1143-1344) and is 12 amino acids shorter. Moreover, the EGF-like domains IIIb of laminin alpha3B chain shows 47.2% homology with its counterpart in laminin alpha1 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 alpha1 chain (between positions 981 and 1142) (Sasaki et al., 1988). Domains IIIa of laminin chains alpha3B and alpha1 are 39.5% homologous. In the alpha3 chain, domain IIIa retains the four EGFs that constitute domain IIIa in the alpha1 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 alpha3B chain and their sequence homology with the corresponding domains of mouse and Drosophila alpha chains are summarized in Table 1.



The mouse alpha3B chain presents 77% homology to the available sequences of the human alpha3B chain (Fig. 2A). 42 cysteine residues detected in the human alpha3B 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 alpha3B 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 alpha3B 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 alpha3B chain (residues 369-371) (Ryan et al., 1994). In human and mouse alpha3B chain, 11 glycosylation sites are in conserved positions (Fig. 2A); however, the three glycosylation sites found at positions 1209, 1326, and 1668 in the alpha3EpB chain (Ryan et al., 1994) are substituted by the three glycosylation sites at positions 912, 1398, and 1596 in the mouse sequence.

Domain Structure of Laminin alpha3A Chain Isoform

The mouse laminin isoforms alpha3A and alpha3B present totally divergent 5`-ends and share identical amino acid sequences downstream of position 901 of the alpha3B cDNA (Fig. 2A). The 5`-end region of laminin alpha3A chain is encoded by the cDNA clone PR6 and comprises 43 NH(2)-terminal amino acids specific to this chain that display 67.4% identity to the homologous 5`-amino acid sequence of the human alpha3A counterpart (Fig. 2B). The Gln residue at position 44 (Gln-901 in laminin alpha3B chain) is the first amino acid common to both alpha3A and alpha3B isoforms (Fig. 2A). The full-length alpha3A cDNA (5563 nucleotides) comprises a 5`-untranslated region of 62 nucleotides and an ORF (5133 nucleotides), beginning with a Met codon surrounded by sequences fitting the eukaryotic translation start sites (Kozak, 1991). The initiation methionine precedes an appropriate signal sequence of 17 amino acids with consensus cleavage site following residue Glu-18 (Von Heijne, 1986) (Fig. 2B, arrow). The ORF encodes a protein of 1711 amino acids with nine consensus sites for N-linked glycosylation. The mature peptide has a predicted mass of 186,230 Da. The mass of the glycosylated peptide is estimated at 214,000 Da.

Differential Expression of Laminin Variants alpha3A and alpha3B

Since previous studies on the tissue distribution of the murine laminin alpha3 chain were performed using probes specific to the peptide COOH-terminal domains common to both alpha3A and alpha3B variants (Aberdam et al., 1994), we investigated whether cDNA probes for the distinct amino-terminal domains of the alpha3 chain isoforms detected expression patterns specific to each polypeptide. We first assessed the expression rate of the alpha3A and alpha3B transcripts by Northern blot analysis of mRNA purified from mouse lung and skin epithelial cells. Hybridization performed with the radioactive cDNA M100, which encodes for the rod-like domain common to both alpha3A and alpha3B chains, identified faint bands corresponding to transcripts with a size ranging between 5.5 kb in skin cells and 8.0 kb detected only in the lung (Fig. 3A). Using cDNA M22 as a radioactive probe specific for the alpha3B chain, a unique 8.0-kb band was detected in lung extracts (Fig. 3B), whereas using cDNA PR6, which encodes the amino-terminal domain of alpha3A, only the 5.5-kb band was specifically observed in epidermal cells (Fig. 3C). These results therefore indicated that some epithelia may express only one of the laminin alpha3 chain isoforms.


Figure 3: Expression of laminin alpha3 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 alpha3A and alpha3B chains (panelA), M22, which is specific to the transcript alpha3B (panelB), and PR6H, which is specific to the alpha3A transcript (panelC).



To verify this possibility, we further investigated the expression of laminin alpha3A and alpha3B 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 alpha3 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 alpha3A 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 alpha3B 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 alpha3A and alpha3B chains, respectively. These results are therefore consistent with a cell type-specific expression of laminin alpha3A and alpha3B 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.



Focal Distribution of Laminin alpha3A and alpha3B Chain Isoforms

We then determined the tissue distribution of laminin alpha3A and alpha3B chains by in situ hybridization on mouse tissues using RNA probes specific for each isoform. According to our previous results (Aberdam et al., 1994), transcripts for both laminin alpha3A and alpha3B chains were detected in the basal membrane of the upper alimentary tract and urinary and nasal epithelia. The alpha3A chain appeared prominently expressed in the skin and, specifically, in hair follicles (Fig. 5A) and developing neurons of the trigeminal ganglion (13.5 days postcoitum) (Table 2). Strong expression of the alpha3B chain was detected in the salivary glands and teeth, where the presence of alpha3A transcripts was also noticed. Conversely, alpha3B transcripts were exclusively found in the bronchi and alveoli, in the stomach and intestinal crypts, in the whisker pads (Fig. 5D), and in the central nervous system (Table 2). In the brain, strong hybridization signals were seen in the telencephalic neuroectoderm (Fig. 5F) and a transient (13.5 days postcoitum), and focalized expression was also observed in the thalamus, the Rathke's pouch, and the periventricular subependymal germinal layer (Fig. 5F).


Figure 5: In situ hybridization of mouse fetal tissue sections with laminin alpha3A (A, C, and E) and alpha3B (B, D, and F) antisense probes. Skin sections show the dermo-epidermal junction and hair follicles with alpha3A probe (A) and negative staining with alpha3B probe (B). Conversely, probe alpha3B stains whisker pads (D), which are not labeled with alpha3A probe (C). Sagittal section of a 13.5-day embryo head revealing negative staining with alpha3A probe (E) and strong staining with alpha3B 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.






DISCUSSION

In the present study, we relate the cloning of cDNAs coding for the full-length alpha3A and alpha3B isoforms of mouse laminin alpha3 chain, and we demonstrate the tissue-specific distribution of these laminin variants.

Sequence data reveal that the alpha3B 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 alpha3A counterpart (200 kDa). These observations are concordant with previous results reporting that human laminin alpha3A 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 alpha3B 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 alpha1 chain, the short arm of mouse laminin alpha3B chain presents no homology with laminin alpha1 and alpha2 chains. The most amino-terminal domain of the polypeptide displays a weak sequence similarity with domain IV" of Drosophila laminin alpha chain, which has been suggested to arise from the fusion of a duplicated domain IV` of laminin beta1 chain (Kusche-Gullberg et al., 1992). However, in this, the two laminin alpha chains differ because no homology is found between the alpha3B chain and laminin beta 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 alpha3B chain short arm. It has thus been suggested that the alpha3B transcript corresponds to the alpha chain polypeptide of laminin 6 (K-laminin) (Ryan et al., 1994). However, lines of evidence suggest that the alpha chains of these two laminins are distinct isoforms. First, the alpha 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 alpha3B is inconsistent with the estimated mass of the alpha chain of laminin 6 (190 kDa) (Marinkovich et al., 1992b). No evidence for processing of the alpha 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 alpha3A and alpha3B transcripts (Vidal et al., 1995). Therefore, detection of the 300-kDa polypeptide corresponding to the alpha3B transcript in lung epithelia, where the laminin alpha3A chain is not detected, supports the assumption of the existence, and the coexistence in some epithelia, of laminin 5 isoforms harboring distinct alpha3 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 alpha6beta4, 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 alpha3B chain to associate with morphological structures distinct from hemidesmosomes via the globular domains of the short arm of the alpha3B chain. This would be consistent with the detection of laminin alpha3B chain in brain tissues where laminin 5 may assume roles other than epithelial adhesion.

The intense expression of the alpha3B 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 alpha1 chain harbors the peptide SIKVAV that mediates cell attachment, migration, and neurite outgrowth (Tashiro et al., 1989). Since in the mouse laminin alpha3 chain the peptide is not conserved, the sequence divergence within this area may reflect a functional difference in the laminin alpha3 chain. The murine laminin alpha3A 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 beta2 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 alpha3 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 alpha3 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 alpha3 chain may contribute to regulate diverse functions of laminin 5. Cloning of the cDNAs for the murine laminin alpha3 chains sets the stage for experiments on gene disruption in mice embryonic stem cells for the analysis of the specific role of the laminin alpha3A and alpha3B chain isoforms.


FOOTNOTES

*
This work was supported by grants from INSERM-CNAMTS, Fondation pour la Recherche Médicale (France), DEBRA Foundation (UK), Groupement de Recherche et d'Etude sur le Génome (GIP-GREG), Association Française contre les Myopathies, and Association pour la Recherche sur le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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].

§
Contributed equally to this work.

Recipient of an E.E.C. Commission.

**
To whom correspondence should be addressed. Tel.: 33-93-37-76-48; Fax: 33-93-81-14-04.

(^1)
The abbreviations used are: kb, kilobase(s); PCR, polymerase chain reaction; 5`-RACE, 5`-rapid amplification of cDNA ends; EGF, epidermal growth factor; ORF, open reading frame.


ACKNOWLEDGEMENTS

We thank Dr. S. Marino, A. Spadafora, and Z. Djabari for expert assistance and C. Minghelli for artwork.


REFERENCES

  1. Aberdam, D., Aguzzi, A., Baudoin, C., Galliano, M. F., Ortonne, J. P., and Meneguzzi, G. (1994) Cell Adhesion Commun. 2,115-129 [Medline] [Order article via Infotrieve]
  2. Baudoin, C., Miquel, C., Blanchet-Bardon, C., Gambini, C., Meneguzzi, G., and Ortonne, J. P. (1994) J. Clin. Invest. 93,862-869 [Medline] [Order article via Infotrieve]
  3. Beck, K., Hunter, I., and Engel, J. (1990) FASEB J. 4,148-160 [Abstract/Free Full Text]
  4. Burgeson, R. E., Chiquet, M., Deutzmann, R., Ekblom, P., Engel, J., Kleinman, H., Martin, G. R., Meneguzzi, G., Paulsson, M., Sanes, J., Timpl, R., Tryggvason, K., Yamada, Y., and Yurchenco, P. D. (1994) Matrix Biol. 14,209-211 [CrossRef][Medline] [Order article via Infotrieve]
  5. Carter, W. G., Kaur, P., Gil, S. G., Gahr, P. J., and Wayner, E. A. (1990) J. Cell Biol. 111,3141-3154 [Abstract]
  6. Carter, W. G., Ryan, M. C., and Gahr, P. J. (1991) Cell 65,599-610 [Medline] [Order article via Infotrieve]
  7. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162,156-159 [CrossRef][Medline] [Order article via Infotrieve]
  8. Gerecke, D., Wagman, D. W., Champliaud, M. F., and Burgeson, R. E. (1994) J. Biol. Chem. 269,11073-11080 [Abstract/Free Full Text]
  9. Hunter, D. D., Porter, B. E., Bulock, J. W., Adams, S. P., Merlie, J. P., and Sanes, J. R. (1989) Cell 59,905-913 [Medline] [Order article via Infotrieve]
  10. Jones, J. C. R., Asmuth, J., Baker, S. E., Langhofer, M., Roth, S. I., and Hopkinson, S. B. (1994) Exp. Cell Res. 213,1-11 [CrossRef][Medline] [Order article via Infotrieve]
  11. Kivirikko, S., McGrath, J., Baudoin, C., Aberdam, D., Ciatti, S., Dunnill, M. G. S., McMillan, J. R., Eady, R. A. J., Ortonne, J. P., Meneguzzi, G., Uitto, J., and Christiano, A. M. (1995) Hum. Mol. Genet. 4,959-962 [Abstract]
  12. Kozak, M. (1991) J. Cell Biol. 115,887-903 [Abstract]
  13. Kusche-Gullberg, M., Garrison, K., Mackrell, A. J., Fessler, L. I., and Fessler, J. H. (1992) EMBO J. 11,4519-4527 [Abstract]
  14. Marinkovich, M. P., Lunstrum, G. P., Keene, D. R., and Burgeson, R. E. (1992a) J. Cell Biol. 119,695-703 [Abstract]
  15. Marinkovich, M. P., Lunstrum, G. P., and Burgeson, R. E. (1992b) J. Biol. Chem. 267,17900-17906 [Abstract/Free Full Text]
  16. Rousselle, P., Lunstrum, G. P., Keene, D. R., and Burgeson, R. E. (1991) J. Cell Biol. 114,567-576 [Abstract]
  17. Ryan, M. C., Tizard, R., VanDevanter, D. R., and Carter, W. G. (1994) J. Biol. Chem. 269,22779-22787 [Abstract/Free Full Text]
  18. Sanes, J. R., Hunter, G. G., Green, T. L., and Merlie, J. P. (1990) Cold Spring Harbor Symp. Quant. Biol. 55,429-430
  19. Sasaki, M., Kleinman, H. K., Huber, H., Deutzmann, R., and Yamada, Y. (1988) J. Biol. Chem. 263,16536-16544 [Abstract/Free Full Text]
  20. Schaeren-Wiemers, N., and Gerfin-Moser, A. (1993) Histochemistry 100,431-440 [Medline] [Order article via Infotrieve]
  21. Sonnenberg, A., Calafat, J., Janssen, H., Daamsn, H., van der Raaij-Helmer, L. M. H., Falcioni, R., Kennel, S. J., Aplin, J. D., Baker, J., Loizidou, M., and Garrod, D. (1991) J. Cell Biol. 113,907-917 [Abstract]
  22. Tashiro, K., Sephel, G. C., Weeks, B., Sasaki, M., Martin, G. R., Kleinman, H. K., and Yamada, Y. (1989) J. Biol. Chem. 264,16174-16182 [Abstract/Free Full Text]
  23. Timpl, R., Rhode, H., Robey, P. G., Renard, S. I., Foidart, J. M., and Martin, G. R. (1979) J. Biol. Chem. 254,9933-9937 [Abstract]
  24. Tryggvason, K. (1993) Curr. Opin. Cell Biol. 5,877-882 [Medline] [Order article via Infotrieve]
  25. Vailly, J., Verrando, P., Champliaud, M. F., Gerecke, D., Wagman, D. W., Baudoin, C., Aberdam, D., Burgeson, R., Bauer, E., and Ortonne, J. P. (1994) Eur. J. Biochem. 219,209-218 [Abstract]
  26. Verrando, P., Hsi, B. L., Yeh, C. J., Pisani, A., Seryeis, N., and Ortonne, J. P. (1987) Exp. Cell Res. 170,116-128 [Medline] [Order article via Infotrieve]
  27. Verrando, P., Blanchet-Bardon, C., Pisani, A., Thomas, L., Cambazard, F., Eady, R. A. J., Schofield, O., and Ortonne, J. P. (1991) Lab. Invest. 64,85-92 [Medline] [Order article via Infotrieve]
  28. Verrando, P., Schofield, O., Ishida-Yamamoto, A., Aberdam, D., Partouche, O., Eady, R. A. J., and Ortonne, J. P. (1993) J. Invest. Dermatol. 101,738-743 [Abstract]
  29. Vidal, F., Baudoin, C., Miquel, C., Galliano, M. F., Christiano, A. M., Uitto, J., Ortonne, J. P., and Meneguzzi, G. (1995) Genomics , in press
  30. Von Heijne, G. (1986) Nucleic Acids Res. 14,4683-4690 [Abstract]
  31. Vuolteenaho, R., Nissinen, M., Sainio, K., Byers, M., Eddy, R., Hirvonen, H., Shows, T. B., Sariola, H., Engvall, E., and Tryggvason, K. (1994) J. Cell Biol. 124,381-394 [Abstract]
  32. Wewer, U. M., Gerecke, D. R., Durkin, M. E., Kurtz, K. S., Mattei, M. G., Champliaud, M. F., Burgeson, R. E., and Albrechtsen, R. (1994) Genomics 24,243-252 [CrossRef][Medline] [Order article via Infotrieve]
  33. Yuspa, S. H., Hawley-Nelson, P., Koehler, B., and Stanley, J. R. (1980) Cancer Res. 40,4694-4703 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.