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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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Biochemical identification of laminins-8, -9, -10, and -11.  (A) Detection of laminin complexes containing α4 and α5. Crude  membrane preparations from adult lung and kidney and purified  laminin-1 were solubilized without reducing agent and fractionated by SDS-PAGE on 3.5% polyacrylamide gels. Proteins were  transferred to nitrocellulose and probed with nonimmune (n.i.)  serum (lanes 2, 6, and 10) or with antibodies to laminin-1 (lanes 1,  5, and 9), α4 (lanes 3, 7, and 11), or α5 (lanes 4, 8, and 12). The  size of the laminin-1 trimer (∼800 kD) is indicated. Lung contained  at least two complexes containing α4 (lane 3), while kidney contained multiple α5-positive complexes (lane 8). Laminin-1 contained little or no detectable α4 or α5. (B) Identification of laminin trimers from adult lung. Laminins were solubilized, partially  purified, and then immunoprecipitated with antibodies to laminin  β1 (lanes 1–5), β2 (lanes 6–10), γ1 (lanes 11–13), or without primary antibody (lanes 14–18). The precipitates were then fractionated on nonreducing gels and probed without primary antibody  (lanes 1, 6, 11, and 14), or with antibodies to laminins β2 (lanes 2,  7, and 15), α4 (lanes 3, 8, 12, and 16), α5 (lanes 4, 9, 13, and 17),  and γ1 (lanes 5, 10, and 18). Four novel laminin trimers were  identified: laminin-8 (α4β1γ1), laminin-9 (α4β2γ1), laminin-10  (α5β1γ1), and laminin-11 (α5β2γ1).
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Figure 10: Biochemical identification of laminins-8, -9, -10, and -11. (A) Detection of laminin complexes containing α4 and α5. Crude membrane preparations from adult lung and kidney and purified laminin-1 were solubilized without reducing agent and fractionated by SDS-PAGE on 3.5% polyacrylamide gels. Proteins were transferred to nitrocellulose and probed with nonimmune (n.i.) serum (lanes 2, 6, and 10) or with antibodies to laminin-1 (lanes 1, 5, and 9), α4 (lanes 3, 7, and 11), or α5 (lanes 4, 8, and 12). The size of the laminin-1 trimer (∼800 kD) is indicated. Lung contained at least two complexes containing α4 (lane 3), while kidney contained multiple α5-positive complexes (lane 8). Laminin-1 contained little or no detectable α4 or α5. (B) Identification of laminin trimers from adult lung. Laminins were solubilized, partially purified, and then immunoprecipitated with antibodies to laminin β1 (lanes 1–5), β2 (lanes 6–10), γ1 (lanes 11–13), or without primary antibody (lanes 14–18). The precipitates were then fractionated on nonreducing gels and probed without primary antibody (lanes 1, 6, 11, and 14), or with antibodies to laminins β2 (lanes 2, 7, and 15), α4 (lanes 3, 8, 12, and 16), α5 (lanes 4, 9, 13, and 17), and γ1 (lanes 5, 10, and 18). Four novel laminin trimers were identified: laminin-8 (α4β1γ1), laminin-9 (α4β2γ1), laminin-10 (α5β1γ1), and laminin-11 (α5β2γ1).

Mentions: Current understanding of the structure and function of BLs is based on the assumption that all laminins are heterotrimers of α, β, and γ subunits, covalently joined by disulfide bonds (Burgeson et al., 1994). Such trimeric structures have been demonstrated for the α1–3 chains, which form laminins 1–7 (Table I), but not for α4 and α5. We therefore asked whether the α4 and α5 chains also occur as components of trimers. To this end, we fractionated proteins from lung and kidney on SDS gels under nonreducing conditions such that laminins migrate as trimers, the relative sizes of which depend on the constituent α, β, and γ chains. Nitrocellulose blots were then probed with antibodies to α4 or α5 (Fig. 10 A). Both lung and kidney contained high Mr complexes that reacted with the α4 (Fig. 10 A, lanes 3 and 7) or α5 (lanes 4 and 8) antisera but not with nonimmune serum (lanes 2 and 6). The α4 complexes were ∼500–600 kD, and the α5 complexes were ∼700–800 kD. These values are consistent with Mrs predicted for laminin trimers. None of the α4- or α5-immunoreactive bands (⩽450 kD) that had been seen after reduction (Fig. 5 B) were detectable under these nonreducing conditions, indicating that all of the monomeric species were associated with larger, disulfide-bonded complexes. Moreover, the approximate difference in Mr between the α4- and α5-containing complexes (∼200 kD) is consistent with the difference in Mr between the full-length α4 and α5 chains themselves. An antiserum to laminin-1, which recognizes the α1, β1, and γ1 chains, blotted material at the Mrs of all α4- and α5containing complexes (Fig. 10 A, lanes 1 and 5), whereas anti-α4 and anti-α5 did not recognize laminin-1 (lanes 11 and 12). Together, these results suggest that laminins α4 and α5 occur in heterotrimers with β1 and/or γ1 chains.


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)

Biochemical identification of laminins-8, -9, -10, and -11.  (A) Detection of laminin complexes containing α4 and α5. Crude  membrane preparations from adult lung and kidney and purified  laminin-1 were solubilized without reducing agent and fractionated by SDS-PAGE on 3.5% polyacrylamide gels. Proteins were  transferred to nitrocellulose and probed with nonimmune (n.i.)  serum (lanes 2, 6, and 10) or with antibodies to laminin-1 (lanes 1,  5, and 9), α4 (lanes 3, 7, and 11), or α5 (lanes 4, 8, and 12). The  size of the laminin-1 trimer (∼800 kD) is indicated. Lung contained  at least two complexes containing α4 (lane 3), while kidney contained multiple α5-positive complexes (lane 8). Laminin-1 contained little or no detectable α4 or α5. (B) Identification of laminin trimers from adult lung. Laminins were solubilized, partially  purified, and then immunoprecipitated with antibodies to laminin  β1 (lanes 1–5), β2 (lanes 6–10), γ1 (lanes 11–13), or without primary antibody (lanes 14–18). The precipitates were then fractionated on nonreducing gels and probed without primary antibody  (lanes 1, 6, 11, and 14), or with antibodies to laminins β2 (lanes 2,  7, and 15), α4 (lanes 3, 8, 12, and 16), α5 (lanes 4, 9, 13, and 17),  and γ1 (lanes 5, 10, and 18). Four novel laminin trimers were  identified: laminin-8 (α4β1γ1), laminin-9 (α4β2γ1), laminin-10  (α5β1γ1), and laminin-11 (α5β2γ1).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139892&req=5

Figure 10: Biochemical identification of laminins-8, -9, -10, and -11. (A) Detection of laminin complexes containing α4 and α5. Crude membrane preparations from adult lung and kidney and purified laminin-1 were solubilized without reducing agent and fractionated by SDS-PAGE on 3.5% polyacrylamide gels. Proteins were transferred to nitrocellulose and probed with nonimmune (n.i.) serum (lanes 2, 6, and 10) or with antibodies to laminin-1 (lanes 1, 5, and 9), α4 (lanes 3, 7, and 11), or α5 (lanes 4, 8, and 12). The size of the laminin-1 trimer (∼800 kD) is indicated. Lung contained at least two complexes containing α4 (lane 3), while kidney contained multiple α5-positive complexes (lane 8). Laminin-1 contained little or no detectable α4 or α5. (B) Identification of laminin trimers from adult lung. Laminins were solubilized, partially purified, and then immunoprecipitated with antibodies to laminin β1 (lanes 1–5), β2 (lanes 6–10), γ1 (lanes 11–13), or without primary antibody (lanes 14–18). The precipitates were then fractionated on nonreducing gels and probed without primary antibody (lanes 1, 6, 11, and 14), or with antibodies to laminins β2 (lanes 2, 7, and 15), α4 (lanes 3, 8, 12, and 16), α5 (lanes 4, 9, 13, and 17), and γ1 (lanes 5, 10, and 18). Four novel laminin trimers were identified: laminin-8 (α4β1γ1), laminin-9 (α4β2γ1), laminin-10 (α5β1γ1), and laminin-11 (α5β2γ1).
Mentions: Current understanding of the structure and function of BLs is based on the assumption that all laminins are heterotrimers of α, β, and γ subunits, covalently joined by disulfide bonds (Burgeson et al., 1994). Such trimeric structures have been demonstrated for the α1–3 chains, which form laminins 1–7 (Table I), but not for α4 and α5. We therefore asked whether the α4 and α5 chains also occur as components of trimers. To this end, we fractionated proteins from lung and kidney on SDS gels under nonreducing conditions such that laminins migrate as trimers, the relative sizes of which depend on the constituent α, β, and γ chains. Nitrocellulose blots were then probed with antibodies to α4 or α5 (Fig. 10 A). Both lung and kidney contained high Mr complexes that reacted with the α4 (Fig. 10 A, lanes 3 and 7) or α5 (lanes 4 and 8) antisera but not with nonimmune serum (lanes 2 and 6). The α4 complexes were ∼500–600 kD, and the α5 complexes were ∼700–800 kD. These values are consistent with Mrs predicted for laminin trimers. None of the α4- or α5-immunoreactive bands (⩽450 kD) that had been seen after reduction (Fig. 5 B) were detectable under these nonreducing conditions, indicating that all of the monomeric species were associated with larger, disulfide-bonded complexes. Moreover, the approximate difference in Mr between the α4- and α5-containing complexes (∼200 kD) is consistent with the difference in Mr between the full-length α4 and α5 chains themselves. An antiserum to laminin-1, which recognizes the α1, β1, and γ1 chains, blotted material at the Mrs of all α4- and α5containing complexes (Fig. 10 A, lanes 1 and 5), whereas anti-α4 and anti-α5 did not recognize laminin-1 (lanes 11 and 12). Together, these results suggest that laminins α4 and α5 occur in heterotrimers with β1 and/or γ1 chains.

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