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The laminin alpha chains: expression, developmental transitions, and chromosomal locations of alpha1-5, identification of heterotrimeric laminins 8-11, and cloning of a novel alpha3 isoform.

Miner JH, Patton BL, Lentz SI, Gilbert DJ, Snider WD, Jenkins NA, Copeland NG, Sanes JR - J. Cell Biol. (1997)

Bottom Line: Interspecific backcross mapping of the five alpha chain genes revealed that they are distributed on four mouse chromosomes.Finally, we identified a novel full-length alpha3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains.Together, these results reveal remarkable diversity in BL composition and complexity in BL development.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
Laminin trimers composed of alpha, beta, and gamma chains are major components of basal laminae (BLs) throughout the body. To date, three alpha chains (alpha1-3) have been shown to assemble into at least seven heterotrimers (called laminins 1-7). Genes encoding two additional alpha chains (alpha4 and alpha5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant alpha4 and alpha5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that alpha4 and alpha5 assemble into four novel laminin heterotrimers (laminins 8-11: alpha4beta1gamma1, alpha4beta2gamma1, alpha5beta1gamma1, and alpha5beta2gamma1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of alpha1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, alpha4 and alpha5 exhibited the broadest patterns of expression, while expression of alpha1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the alpha chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one alpha chain, all alpha chains were present in multiple BLs, and some BLs contained two or three alpha chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different alpha chains and two different beta chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five alpha chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length alpha3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains. Together, these results reveal remarkable diversity in BL composition and complexity in BL development.

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Nucleotide and deduced amino acid sequences of the  laminin α3B chain, 5′ and NH2-terminal to those reported by  Galliano et al. (1995). Overlapping nucleotides are in lowercase.  The Galliano sequence contains a single deoxyguanosine (parentheses) not found in our sequence, which shifts the reading frame  and leads to an encoded methionine (bottom line, italics). Domains  are marked as in Fig. 1. This was hypothesized to be the initiator  for translation. An adhesive tripeptide sequence, LRE (Hunter  et al., 1989a), is indicated by bullets; another LRE is located in  the G domain (Galliano et al., 1995). These sequence data are  available from GenBank under accession number U88353.
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Figure 2: Nucleotide and deduced amino acid sequences of the laminin α3B chain, 5′ and NH2-terminal to those reported by Galliano et al. (1995). Overlapping nucleotides are in lowercase. The Galliano sequence contains a single deoxyguanosine (parentheses) not found in our sequence, which shifts the reading frame and leads to an encoded methionine (bottom line, italics). Domains are marked as in Fig. 1. This was hypothesized to be the initiator for translation. An adhesive tripeptide sequence, LRE (Hunter et al., 1989a), is indicated by bullets; another LRE is located in the G domain (Galliano et al., 1995). These sequence data are available from GenBank under accession number U88353.

Mentions: The only laminin gene so far shown to encode more than a single polypeptide is the laminin α3 gene. The two known products, α3A and α3B, differ at their amino termini, resulting from alternative splicing and/or alternative promoter usage. The shorter α3A chain was the first to be identified by both immunological methods (Rousselle et al., 1991) and by cDNA cloning (Aberdam et al., 1994b). Subsequently, however, multiple α3 cDNAs were identified in human (Ryan et al., 1994) and mouse (Galliano et al., 1995), suggesting the existence of a second, longer isoform called α3B. Sequence analysis (Galliano et al., 1995) indicated that α3B contains two cysteine-rich (IIIa and IIIb) and two globular (IVa and IV′′) domains in addition to the G and I/II domains, but no domains V or VI. This would make it the sole “mid-sized” α chain. However, we have obtained mouse cDNAs (see Materials and Methods) that extend the previously reported sequence 5′ by ∼2.2 kb (Fig. 2). This novel sequence encodes the NH2-terminal portion of domain IVb, a complete domain V, and what is likely all but a few amino acids of a domain VI. These domains are most similar in sequence and predicted tertiary structure to the analogous domains of laminin α5. For example, the amino acid sequence of domain VI is 74% identical to that of α5, but only 54 and 51% identical to those of α1 and α2, respectively. A probe from the 5′ end of this sequence recognized ∼10-kb bands on Northern blots of mouse RNA from adult brain, lung, and kidney (data not shown). The proposed α3B structure is shown in Fig. 1, indicating its unique NH2 terminus and the COOH terminus it shares with α3A.


The laminin alpha chains: expression, developmental transitions, and chromosomal locations of alpha1-5, identification of heterotrimeric laminins 8-11, and cloning of a novel alpha3 isoform.

Miner JH, Patton BL, Lentz SI, Gilbert DJ, Snider WD, Jenkins NA, Copeland NG, Sanes JR - J. Cell Biol. (1997)

Nucleotide and deduced amino acid sequences of the  laminin α3B chain, 5′ and NH2-terminal to those reported by  Galliano et al. (1995). Overlapping nucleotides are in lowercase.  The Galliano sequence contains a single deoxyguanosine (parentheses) not found in our sequence, which shifts the reading frame  and leads to an encoded methionine (bottom line, italics). Domains  are marked as in Fig. 1. This was hypothesized to be the initiator  for translation. An adhesive tripeptide sequence, LRE (Hunter  et al., 1989a), is indicated by bullets; another LRE is located in  the G domain (Galliano et al., 1995). These sequence data are  available from GenBank under accession number U88353.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139892&req=5

Figure 2: Nucleotide and deduced amino acid sequences of the laminin α3B chain, 5′ and NH2-terminal to those reported by Galliano et al. (1995). Overlapping nucleotides are in lowercase. The Galliano sequence contains a single deoxyguanosine (parentheses) not found in our sequence, which shifts the reading frame and leads to an encoded methionine (bottom line, italics). Domains are marked as in Fig. 1. This was hypothesized to be the initiator for translation. An adhesive tripeptide sequence, LRE (Hunter et al., 1989a), is indicated by bullets; another LRE is located in the G domain (Galliano et al., 1995). These sequence data are available from GenBank under accession number U88353.
Mentions: The only laminin gene so far shown to encode more than a single polypeptide is the laminin α3 gene. The two known products, α3A and α3B, differ at their amino termini, resulting from alternative splicing and/or alternative promoter usage. The shorter α3A chain was the first to be identified by both immunological methods (Rousselle et al., 1991) and by cDNA cloning (Aberdam et al., 1994b). Subsequently, however, multiple α3 cDNAs were identified in human (Ryan et al., 1994) and mouse (Galliano et al., 1995), suggesting the existence of a second, longer isoform called α3B. Sequence analysis (Galliano et al., 1995) indicated that α3B contains two cysteine-rich (IIIa and IIIb) and two globular (IVa and IV′′) domains in addition to the G and I/II domains, but no domains V or VI. This would make it the sole “mid-sized” α chain. However, we have obtained mouse cDNAs (see Materials and Methods) that extend the previously reported sequence 5′ by ∼2.2 kb (Fig. 2). This novel sequence encodes the NH2-terminal portion of domain IVb, a complete domain V, and what is likely all but a few amino acids of a domain VI. These domains are most similar in sequence and predicted tertiary structure to the analogous domains of laminin α5. For example, the amino acid sequence of domain VI is 74% identical to that of α5, but only 54 and 51% identical to those of α1 and α2, respectively. A probe from the 5′ end of this sequence recognized ∼10-kb bands on Northern blots of mouse RNA from adult brain, lung, and kidney (data not shown). The proposed α3B structure is shown in Fig. 1, indicating its unique NH2 terminus and the COOH terminus it shares with α3A.

Bottom Line: Interspecific backcross mapping of the five alpha chain genes revealed that they are distributed on four mouse chromosomes.Finally, we identified a novel full-length alpha3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains.Together, these results reveal remarkable diversity in BL composition and complexity in BL development.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
Laminin trimers composed of alpha, beta, and gamma chains are major components of basal laminae (BLs) throughout the body. To date, three alpha chains (alpha1-3) have been shown to assemble into at least seven heterotrimers (called laminins 1-7). Genes encoding two additional alpha chains (alpha4 and alpha5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant alpha4 and alpha5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that alpha4 and alpha5 assemble into four novel laminin heterotrimers (laminins 8-11: alpha4beta1gamma1, alpha4beta2gamma1, alpha5beta1gamma1, and alpha5beta2gamma1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of alpha1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, alpha4 and alpha5 exhibited the broadest patterns of expression, while expression of alpha1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the alpha chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one alpha chain, all alpha chains were present in multiple BLs, and some BLs contained two or three alpha chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different alpha chains and two different beta chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five alpha chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length alpha3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains. Together, these results reveal remarkable diversity in BL composition and complexity in BL development.

Show MeSH
Related in: MedlinePlus