©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning of a Novel Laminin Chain, 5, and Widespread Expression in Adult Mouse Tissues (*)

(Received for publication, September 27, 1995)

Jeffrey H. Miner (§) Renate M. Lewis Joshua R. Sanes (¶)

From the Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have identified a fifth member of the alpha subfamily of vertebrate laminin chains. Sequence analysis revealed a close relationship of alpha5 to the only known Drosophila alpha chain, suggesting that the ancestral alpha gene was more similar to alpha5 than to alpha1-4. Analysis of RNA expression showed that alpha5 is widely expressed in adult tissues, with highest levels in lung, heart, and kidney. Our results suggest that alpha5 may be a major laminin chain of adult basal laminae.


INTRODUCTION

Laminins are major glycoproteins of basal laminae throughout the vertebrate body. Originally identified as structural components, it is now clear that laminins are also signaling molecules that regulate the proliferation, motility, and differentiation of the cells they contact (1, 2, 3) . The first laminin discovered(4, 5) , now called laminin-1(6) , is a trimer of related A, B1, and B2 chains(7, 8, 9) . The subsequent discovery of the novel laminin chains S-laminin (10) and merosin-M (11) revealed that the laminins comprised a larger gene family than initially envisioned. More recently, four additional chains have been cloned(12, 13, 14, 15, 16, 17, 18, 19) . So far, however, all laminin chains sequenced resemble either A, B1, or B2, and all of the laminins purified are trimers containing an A-like, a B1-like, and a B2-like chain(6, 19, 20, 21, 22) . Based on these findings, a new nomenclature has been adopted in which laminin chains are divided into alpha (A-like), beta (B1-like) and (B2-like) subfamilies; A, M, B1, S, and B2 are now called alpha1, alpha2, beta1, beta2, and 1, respectively(6) .

Consistent with laminin's trimeric structure, all basal laminae characterized to date contain at least one beta and at least one chain(19, 20, 23, 24, 25, 26, 27, 28) . For the alpha chains, on the other hand, the situation is less clear. For example, perineurial basal lamina in peripheral nerve stained poorly with anti-alpha1 and not at all with anti-alpha2(23) . Likewise, in kidney, glomerular basement membrane was alpha2-negative and reacted only moderately well (in human(23) ) or not at all (in mouse(29, 30) ) with anti-alpha1. If all laminins are alpha/beta/ trimers, these results imply that additional laminin alpha chains exist. Indeed, several biochemical studies have provided evidence for an alpha-like laminin chain distinct from alpha1 and alpha2 (31, 32, 33) . The recent discoveries of the alpha3 and alpha4 chains(15, 16, 17, 18) are provocative in this context, but we (^1)and others (16, 18) found little alpha3 or alpha4 in several tissues with alpha1- and alpha2-negative basal laminae.

Accordingly, we undertook a search for additional laminin alpha chains. Using the polymerase chain reaction, we have identified laminin alpha5, a novel murine alpha chain. Sequence analysis reveals that alpha5 is more similar in domain structure to Drosophila laminin A(34, 35) than it is to mammalian alpha1-4; the ancestral vertebrate alpha chain may, therefore, have been more similar to alpha5 that to alpha1-4. RNA analysis demonstrates that alpha5 is widely expressed in adult tissues and thus may be a major laminin alpha chain of adult basal laminae.


MATERIALS AND METHODS

Degenerate primers were designed based on sequences conserved between mouse laminin alpha1 (7) and human laminin alpha2(36) . Reverse transcription-PCR (^2)was performed on RNA from postnatal day 7 mouse kidney using a GeneAmp kit (Perkin-Elmer). One pair of primers from the amino terminus of domain V (sense, 5`-GGNAGTTGYATHTGYTAYGGNC-3` and antisense, 5`-TRCANTGYTCRCARTTDATNCC-3`, where N = A/G/C/T, H = A/T/C, and D = G/A/T) produced a fragment of appropriate length (330 base pairs). The band was excised from agarose (SeaPlaque GTG, FMC Bioproducts, Rockland, ME), reamplified with the same primers, purified with a Wizard PCR Preps kit (Promega Corp., Madison, WI), and incubated with BglI to digest laminin alpha1 products, thereby preventing their further amplification. The remaining full-length product was reamplified and ligated into the pCRII vector (Invitrogen Corp., San Diego, CA).

One resulting clone, DB2, bore an insert related to but distinct from laminins alpha1 and alpha2. The DB2 insert was labeled with [P]dCTP by the random primed DNA labeling kit (Boehringer Mannheim) and used to screen an adult mouse lung oligo(dT)+ random primed ZAP II cDNA library (Stratagene, La Jolla, CA). Subsequent cDNA library screening was performed as a ``walk'' using selected restriction fragments of hybridizing phage to obtain overlapping clones. Clones were sequenced on an ABI 373A DNA sequencer using a Taq DyeDeoxy Terminator cycle sequencing kit (Applied Biosystems Inc., Foster City, CA). All sequences were determined from both strands. Data base homology searches were performed on the BLAST server at the National Center for Biotechnology Information(37) , and sequences were compared using Genetics Computing Group programs(38) .

For Northern analysis, a filter containing poly(A)-selected RNA from several adult mouse tissues (Clontech) was hybridized with a probe comprising nucleotides 7855-9361 of the alpha5 sequence. RNase protection analysis was performed as described previously(24) .


RESULTS AND DISCUSSION

Molecular Cloning of Laminin alpha5

We used PCR to amplify a 334-base pair fragment from mouse kidney that encoded a novel laminin-like sequence. This fragment was used as the starting point for isolation of a series of overlapping cDNA clones that spanned >11 kb. Sequence analysis revealed that the cDNAs encoded a single open reading frame of 3610 amino acids (Fig. 1). The sequence of the predicted protein is related to but clearly distinct from those of previously reported laminin chains(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 34, 35, 36) . As detailed below, the novel sequence is more closely related to laminin alpha chains than to beta or chains. Because four mammalian laminin alpha chains have been described to date(7, 11, 15, 16, 17, 18) , we have named the novel chain laminin alpha5.


Figure 1: Amino acid sequence of the mouse laminin alpha5 chain, deduced from cDNAs. Domain boundaries are noted and the adhesive tripeptide sequences, RGD (40) and LRE(42) , are indicated by bullets. The nucleotide sequence is available from GenBank under accession number U37501.



The first amino acid of the deduced sequence is aspartic acid rather than methionine, indicating that the cDNAs do not reach the 5` end of the coding sequence. Repeated efforts to obtain additional cDNAs failed. Based on homology to other laminin alpha chains, we believe that the mature protein contains 3630 amino acids, of which we have identified 3610.

Comparison with Other Laminin Chains

The laminin alpha5 chain contains eight domains, based on predicted secondary structure and homology to the laminin alpha1 chain (Fig. 2A). Their nomenclature follows that for alpha1, which was in turn designed to maintain consistency with the beta1 and 1 chains(1, 7) . The carboxyl-terminal half of the protein contains a large (100 kDa) globular domain (G) and an alpha-helical segment (I/II). The amino-terminal half contains three cysteine- and glycine-rich regions predicted to form rigid, rodlike structures (IIIa, IIIb, and V) and three smaller globular regions (IVa, IVb, and VI). Five of the domains are characterized by repeating structures: heptad repeats with hydrophobic residues in the first and fourth position of domain I/II; ``EGF-like'' repeats of 50 amino acids each in domains IIIa, IIIb, and V; and five tandem ``G'' repeats of 186 amino acids each in G.


Figure 2: Relationship of alpha5 to other laminin chains. A, domain structure of the known laminin alpha chains. The names of the domains, based on accepted nomenclature(1, 7) , are to the left of alpha5. Percent amino acid identity of individual domains of alpha5 with the corresponding domains of the other alpha chains, determined with the GAP program(38) , is shown to the left of the other alpha chains. Numbers of EGF repeats, rounded to the nearest integer, are indicated within domains III and V. Domain structures of beta1 and 1 are shown to indicate the basis for assigning alpha5 to the alpha subfamily. alphaD, Drosophila A. B, relationships among mammalian and Drosophila alpha chains, based on sequence alignment performed by the PILEUP program(38) . Comparisons were based on domains G-IIIa, which all alpha chains contain. C, evolutionary scheme for vertebrate alpha chains, incorporating comparisons of primary sequences (from B) and predicted secondary structure (from A). In this scheme, the ancestral alpha gene was more similar in domain structure to alpha5 than to alpha1-4. See text for details.



Several features of alpha5 identify it as an alpha chain (Fig. 2A). First, it contains a G domain, which is present in all alpha but no beta or chains. Second, alpha5, like alpha1 and alpha2, contains three sets of EGF-like repeats (IIIa, IIIb, and V) and three globular regions (IVa, IVb, and VI) in its amino-terminal half, whereas no beta or chain has more than two of each. Third, alpha5 lacks the alpha-insert between domains I and II that characterizes beta chains.

Of the four vertebrate laminin alpha chains characterized to date, two (alpha1 and alpha2) contain domains G-VI, whereas the other two (alpha3 and alpha4) are truncated and contain only domains G-IIIa (Fig. 2A). An alternatively spliced product of the alpha3 gene, alpha3B, which contains domains IIIb and IV, has been identified but not yet fully sequenced(12) . In its domain structure, alpha5 is more similar to alpha1 and alpha2 than to alpha3 or alpha4, and it can therefore be classified as a ``full-length'' alpha chain. On the other hand, within the domains shared by all alpha chains, the laminin alpha5 sequence is more similar to alpha3 and alpha4 than to alpha1 or alpha2 (Fig. 2B). Thus, sequence analysis reveals an apparent discrepancy between relationships based on primary and secondary structure.

More surprising is that alpha5 is more similar in domain structure to the only known Drosophila A chain (alphaD(34, 35) ) than it is to any of the vertebrate alpha chains. Both alpha5 and alphaD contain 11 EGF repeats in domain V, 4 in domain IIIb, and 7 in domain IIIa, whereas alpha1 and alpha2 have 4, 9, and 4 repeats in domains V, IIIb, and IIIa, respectively (Fig. 2A). Likewise, domain IVb is much larger in alphaD and alpha5 (558 and 577 amino acids) than in alpha1 or alpha2 (196 amino acids).

Together, these results suggest the evolutionary scheme diagrammed in Fig. 2C. We propose than an ancestral alpha gene, similar in domain structure to alphaD and alpha5, was duplicated early in the vertebrate lineage. One of the daughter genes evolved the alpha1/2 domain structure, perhaps by recombination(39) , and was then duplicated again to generate alpha1 and alpha2. The other product of the original duplication was also reduplicated. One daughter evolved into alpha5 without further rearrangement, while the other suffered truncation and then duplicated yet again to generate alpha3 and alpha4. Although speculative, this scheme accounts for the otherwise puzzling findings that alpha5 resembles alpha1 and alpha2 in secondary structure but is most closely related to alpha3 and alpha4 in primary sequence.

Studies with synthetic peptides have provided evidence for several discrete adhesive sites within laminin alpha chains, although their significance in vivo remains unclear. The tripeptide RGD, which is recognized by several integrins(40) , is present in alpha1, alpha3, and alpha4 but not in alpha2 or alphaD(7, 15, 18, 34, 35, 36, 41) ; it is present twice within the alpha5 sequence (Fig. 1). The tripeptide LRE, a major determinant of a motoneuron-selective adhesive site in beta2(42, 43) , is present in alpha1, alpha3, and alphaD but not in alpha2 or alpha4(7, 15, 18, 34, 35, 36, 41) ; it is present twice in alpha5. Finally, the sequence IKVAV, an adhesive site in alpha1(44) , is replaced by SKVKV in alpha5.

Expression of Laminin alpha5

RNA from a panel of adult mouse tissues was subjected to Northern analysis with an alpha5 cDNA. High levels of alpha5 mRNA were detected in heart, lung, and kidney (Fig. 3A). Longer exposures revealed lower but significant levels of alpha5 mRNA in brain, muscle, and testis (Fig. 3B). In all of these tissues, an RNA species of 12 kb was detected; an additional RNA of 9 kb, visible only in testis, would be unable to encode full-length alpha5. A more sensitive RNase protection assay revealed significant levels of alpha5 RNA in liver, as well as in gut and skin (not shown). Moreover, RNase protection and in situ hybridization analyses indicated that alpha5 is expressed in many tissues by embryonic day 11. (^3)Thus, the alpha5 gene is widely expressed.


Figure 3: Northern analysis of laminin alpha5 RNA from adult mouse tissues. Short (15 h (A)) and long (52 h (B)) exposures of a single blot (with an intensifying screen) are shown.



The broad distribution of alpha5 RNA in adult tissues stands in marked contrast to the restricted patterns of expression of the alpha1, alpha3, and alpha4 genes(16, 18, 30, 41) . (^4)Laminin alpha2 RNA is widely distributed in adult tissues but is predominantly expressed by mesenchymal or mesodermal cells(36) . Taken together, these results suggest that laminin alpha5 is a major alpha chain of adult epithelial and/or endothelial basal laminae. Moreover, it seems possible that reports of alpha1-like immunoreactivity in tissues such as kidney, lung, and muscle(20, 23, 25, 26, 27, 28) , which contain little alpha1 RNA, reflect cross-reactivity of anti-alpha1 antibodies with alpha5. We are currently preparing monospecific antibodies to evaluate this possibility.


FOOTNOTES

*
This work was supported by a National Institutes of Health grant. 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) U37501[GenBank].

§
Supported by a fellowship from the Damon Runyon-Walter Winchell Cancer Research Fund.

To whom correspondence should be addressed: Dept. of Anatomy and Neurobiology, Washington University School of Medicine, 660 South Euclid Ave., Box 8108, St. Louis, MO 63110. Tel.: 314-362-2507; Fax: 314-747-1150; sanesj@thalamus.wustl.edu.

(^1)
J. H. Miner, J. R. Sanes, and D. Aberdam, unpublished data.

(^2)
The abbreviations used are: PCR, polymerase chain reaction; kb, kilobase(s); EGF, epidermal growth factor.

(^3)
J. H. Miner, S. I. Lentz, W. D. Snider, and J. R. Sanes, manuscript in preparation.

(^4)
J. H. Miner, unpublished results.


ACKNOWLEDGEMENTS

We thank Guoping Feng and Jacqueline Mudd for advice and assistance.


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