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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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Identification of laminin α4 and α5 proteins in lung and  kidney. (A) Characterization of antisera. The α4 and α5 fusion  proteins used to immunize rabbits were fractionated by SDSPAGE on 12% gels and transferred to blots. Strips were probed  either with no primary antibody (lanes 1 and 4), with the anti-α4  antiserum (lanes 2 and 5), or with the anti-α5 antiserum (lanes 3  and 6). Each antiserum specifically recognized its cognate immunogen. (B) Solubilized and reduced crude membranes from adult  rat lung and kidney and purified laminin-1 were fractionated on  7% gels and transferred to blots. Strips were probed either with  anti–laminin-1 (lanes 1, 6, and 10), nonimmune (lanes 2, 7, and  11), antilaminin α4 (lanes 3, 8, and 12), or anti-laminin α5 (lanes  4, 5, 9, and 13). The anti-α4 serum recognized a protein of ∼180  kD in lung and kidney. The α5 antiserum recognized several  bands in lung and kidney (lanes 4 and 9), the largest of which,  ∼450 kD, was observed only after long exposures (lane 5, arrowheads). Neither serum recognized laminin α1 (lanes 12 and 13).  n.i., nonimmune serum; x, nonspecific bands seen in all lung lanes  with long exposures.
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Figure 5: Identification of laminin α4 and α5 proteins in lung and kidney. (A) Characterization of antisera. The α4 and α5 fusion proteins used to immunize rabbits were fractionated by SDSPAGE on 12% gels and transferred to blots. Strips were probed either with no primary antibody (lanes 1 and 4), with the anti-α4 antiserum (lanes 2 and 5), or with the anti-α5 antiserum (lanes 3 and 6). Each antiserum specifically recognized its cognate immunogen. (B) Solubilized and reduced crude membranes from adult rat lung and kidney and purified laminin-1 were fractionated on 7% gels and transferred to blots. Strips were probed either with anti–laminin-1 (lanes 1, 6, and 10), nonimmune (lanes 2, 7, and 11), antilaminin α4 (lanes 3, 8, and 12), or anti-laminin α5 (lanes 4, 5, 9, and 13). The anti-α4 serum recognized a protein of ∼180 kD in lung and kidney. The α5 antiserum recognized several bands in lung and kidney (lanes 4 and 9), the largest of which, ∼450 kD, was observed only after long exposures (lane 5, arrowheads). Neither serum recognized laminin α1 (lanes 12 and 13). n.i., nonimmune serum; x, nonspecific bands seen in all lung lanes with long exposures.

Mentions: The α1–3 chains were first identified by biochemical and immunochemical methods (Timpl et al., 1979; Leivo and Engvall, 1988; Rousselle et al., 1991; Carter et al., 1991; Verrando et al., 1992). In contrast, α4 and α5 were identified as cDNAs by molecular cloning (Richards et al., 1994; Iivanainen et al., 1995; Miner et al., 1995). Sequence analysis implies that the α4 and α5 cDNAs encode laminin-like proteins, but it is crucial to demonstrate this directly. To this end, we used α4 and α5 cDNAs to produce recombinant proteins in bacteria, and then used the proteins to generate antisera in rabbits. Each antiserum specifically recognized its immunogen on Western blots (Fig. 5 A), and immunoreactivity was removed by incubation with the corresponding fusion protein. Since α3B and α5 sequences are closely related (see above), we also tested anti-α5 on a recombinant fragment from the corresponding domains of α3B. No cross-reaction was detected (data not shown).


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)

Identification of laminin α4 and α5 proteins in lung and  kidney. (A) Characterization of antisera. The α4 and α5 fusion  proteins used to immunize rabbits were fractionated by SDSPAGE on 12% gels and transferred to blots. Strips were probed  either with no primary antibody (lanes 1 and 4), with the anti-α4  antiserum (lanes 2 and 5), or with the anti-α5 antiserum (lanes 3  and 6). Each antiserum specifically recognized its cognate immunogen. (B) Solubilized and reduced crude membranes from adult  rat lung and kidney and purified laminin-1 were fractionated on  7% gels and transferred to blots. Strips were probed either with  anti–laminin-1 (lanes 1, 6, and 10), nonimmune (lanes 2, 7, and  11), antilaminin α4 (lanes 3, 8, and 12), or anti-laminin α5 (lanes  4, 5, 9, and 13). The anti-α4 serum recognized a protein of ∼180  kD in lung and kidney. The α5 antiserum recognized several  bands in lung and kidney (lanes 4 and 9), the largest of which,  ∼450 kD, was observed only after long exposures (lane 5, arrowheads). Neither serum recognized laminin α1 (lanes 12 and 13).  n.i., nonimmune serum; x, nonspecific bands seen in all lung lanes  with long exposures.
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Figure 5: Identification of laminin α4 and α5 proteins in lung and kidney. (A) Characterization of antisera. The α4 and α5 fusion proteins used to immunize rabbits were fractionated by SDSPAGE on 12% gels and transferred to blots. Strips were probed either with no primary antibody (lanes 1 and 4), with the anti-α4 antiserum (lanes 2 and 5), or with the anti-α5 antiserum (lanes 3 and 6). Each antiserum specifically recognized its cognate immunogen. (B) Solubilized and reduced crude membranes from adult rat lung and kidney and purified laminin-1 were fractionated on 7% gels and transferred to blots. Strips were probed either with anti–laminin-1 (lanes 1, 6, and 10), nonimmune (lanes 2, 7, and 11), antilaminin α4 (lanes 3, 8, and 12), or anti-laminin α5 (lanes 4, 5, 9, and 13). The anti-α4 serum recognized a protein of ∼180 kD in lung and kidney. The α5 antiserum recognized several bands in lung and kidney (lanes 4 and 9), the largest of which, ∼450 kD, was observed only after long exposures (lane 5, arrowheads). Neither serum recognized laminin α1 (lanes 12 and 13). n.i., nonimmune serum; x, nonspecific bands seen in all lung lanes with long exposures.
Mentions: The α1–3 chains were first identified by biochemical and immunochemical methods (Timpl et al., 1979; Leivo and Engvall, 1988; Rousselle et al., 1991; Carter et al., 1991; Verrando et al., 1992). In contrast, α4 and α5 were identified as cDNAs by molecular cloning (Richards et al., 1994; Iivanainen et al., 1995; Miner et al., 1995). Sequence analysis implies that the α4 and α5 cDNAs encode laminin-like proteins, but it is crucial to demonstrate this directly. To this end, we used α4 and α5 cDNAs to produce recombinant proteins in bacteria, and then used the proteins to generate antisera in rabbits. Each antiserum specifically recognized its immunogen on Western blots (Fig. 5 A), and immunoreactivity was removed by incubation with the corresponding fusion protein. Since α3B and α5 sequences are closely related (see above), we also tested anti-α5 on a recombinant fragment from the corresponding domains of α3B. No cross-reaction was detected (data not shown).

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