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

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.

Figure 1

The laminin α chain subfamily. Numbering of domains is based on accepted nomenclature (Sasaki et al., 1988; Engvall and Wewer, 1996). Numbers of laminin-type cysteine-rich (EGFlike) repeats, rounded to the nearest integer, are indicated within each domain IIIa, IIIb, or V. Note that laminins α3A and α3B share domains G, I/II, and IIIa but have distinct NH₂ termini.

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.

Figure 3

Ribonuclease protection analysis of laminin α chain expression in (A) adult and (B) E17.5 mouse tissues. A probe for elongation factor 1α was used to control for the amount of input RNA in both embryos (B) and adults (not shown). Each α chain is expressed in a distinct pattern in the adult and, in general, these patterns are established by birth. The α5 chain is the most highly expressed, and α1 is the most restricted. B, brain; I, intestine; H, heart; K, kidney; Li, liver; Lu, lung; M, skeletal muscle; S, skin; P, placenta; Y, yeast RNA. The sample of E17.5 placental RNA was included in the panel of adult RNAs to allow comparison between experiments. nd, not done.

Figure 4

In situ hybridization of laminin α chain probes to E15.5 embryo parasagittal sections. (a) α1, (b) α2, (c) α3, (d) α4, and (e) α5. α1 shows restricted expression in kidney and meninges (arrowheads); α2 and α4 show widespread expression in mesenchymal cells and derivatives as well as in dorsal root ganglia (arrowheads); and α3 and α5 transcripts localize primarily to epithelia. (f–h) High power views of (f) α3, (g) α4, and (h) α5 expression in lung. α3 and α5 are concentrated in the epithelial lung buds, and α4 to the mesenchyme. H, heart; K, kidney; L, lung; SG, salivary gland; T, tongue (muscle). Bars: (d) 1 mm; (h) 50 μm.

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.

Figure 6

Immunohistochemical localization of laminin α chains in adult mouse kidney (a–e), heart (f–j), and lung (k–o). All five α chains were present in adult BLs, but each chain was distributed in a distinct pattern. α1 was found only in kidney mesangium and in a subset of tubular BLs (primarily proximal) (a). α2 was present in mesangium (b), in a subset of corticomedullary tubular BLs (b, inset), and in cardiomyocyte BLs (g). α3 was detected in kidney papillary BL (c) and in lung alveolar BL (m). α4 was absent from renal cortex (d) but found in capillaries of both the renal medulla (not shown) and heart (i), and in alveolar BLs in lung (n). α5 showed the most widespread expression: in all kidney BLs—glomerular, tubular, and arterial (e); in heart blood vessels and in some cardiomyocyte BLs (j); and in lung alveolar BL (o). G, glomerulus; M, mesangium; bv, blood vessel. Bar, 50 μm.

Figure 7

Schematic summary of kidney development, and expression patterns of the laminin α chains in various nephron segment BLs. The laminin α and β chains expressed in the developing glomerular BL (GBM) and its progenitors are boxed. See text for details.

Figure 8

Immunohistochemical analysis of laminins α1, α4, α5, and γ1 in developing kidney shows the dynamic pattern of α chain accumulation depicted schematically in Fig. 7. All sections are from P1 mouse kidney except c, which is from E15.5. b′, c, d′, f′, g, and h′ are double exposures of doubly labeled sections; antibodies listed first and second are shown in green and red, respectively, and regions of overlap are indicated by yellow and light orange. Single exposure companions are shown in b (for b′), d (for d′), f (for f′), and h (for h′). U, ureteric bud; V, vesicle; C, comma-shaped structure; S, S-shaped structure; CL, capillary loop; mG, maturing glomerulus; bv, blood vessel. (Arrows) Progenitors of glomerular BL. Bar, 50 μm.

Figure 9

In situ hybridization of laminin α chain probes to P1 kidney sections. α1 was observed in primitive structures in the cortex and in some tubules (a). α2 was absent from the cortical structures but distributed diffusely in the interior (b). α4 was detected primarily in clusters at the cortex (vesicle and comma stage nephrons) but also diffusely in the medulla (d). α5 was mainly in the collecting ducts, but there were also grain clusters in the inner cortex and in the medulla (e). α3 was absent (c), and a sense control was negative (f). These patterns suggest that the developmental transitions demonstrated immunohistochemically in Figs. 7 and 8 reflect, in part, developmental transitions in α chain gene expression. The cortical surface of the kidney is outlined in white. Insets in a–e show higher power views of the cortex. Bars: (f) 0.5 mm; (a, inset) 0.1 mm.

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).

Figure 11

Chromosomal locations of Lama loci in the mouse genome, as determined from interspecific backcross analysis. The number of recombinant N₂ animals is presented over the total number of N₂ animals typed to the left of the chromosome maps between each pair of loci. The recombination frequencies, expressed as genetic distance in centimorgans (± one standard error) are also shown. The upper 95% confidence limit of the recombination distance is given in parentheses when no recombinants were found between loci. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns. The positions of loci on human chromosomes, where known, are shown to the right of the chromosome maps. (Asterisk) The human LAMA5 gene is predicted to reside on chromosome 20q13. References for the human map positions of loci cited in this study can be obtained from GDB (Genome Data Base), a computerized database of human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, MD).