Presence of Laminin α5 Chain and Lack of Laminin α1 Chain during Human Muscle Development and in Muscular Dystrophies*

There is currently a great interest in identifying laminin isoforms expressed in developing and regenerating skeletal muscle. Laminin α1 has been reported to localize to human fetal muscle and to be induced in muscular dystrophies based on immunohistochemistry with the monoclonal antibody 4C7, suggested to recognize the human laminin α1 chain. Nevertheless, there seems to be no expression of laminin α1 protein or mRNA in developing or dystrophic mouse skeletal muscle fibers. To address the discrepancy between the results obtained in developing and dystrophic human and mouse muscle we expressed the E3 domain of human laminin α1 chain as a recombinant protein and made antibodies specific for human laminin α1 chain (anti-hLN-α1G4/G5). We also made antibodies to the human laminin α5 chain purified from placenta. In the present report we show that hLN-α1G4/G5 antibodies react with a 400-kDa laminin α1 chain and that 4C7 reacts with a 380-kDa laminin α5 chain. Immunohistochemistry with the hLN-α1G4/G5 antibody and 4C7 revealed that the two antibodies stained human kidney, developing and dystrophic muscle in distinct patterns. Our data indicate that the previously reported expression patterns in developing, adult, and dystrophic human muscle tissues with 4C7 should be re-interpreted as an expression of laminin α5 chain. Our data are also consistent with earlier work in mouse, indicating that laminin α1 is largely an epithelial laminin chain not present in developing or dystrophic muscle fibers.

There is currently a great interest in identifying laminin isoforms expressed in developing and regenerating skeletal muscle. Laminin ␣1 has been reported to localize to human fetal muscle and to be induced in muscular dystrophies based on immunohistochemistry with the monoclonal antibody 4C7, suggested to recognize the human laminin ␣1 chain. Nevertheless, there seems to be no expression of laminin ␣1 protein or mRNA in developing or dystrophic mouse skeletal muscle fibers. To address the discrepancy between the results obtained in developing and dystrophic human and mouse muscle we expressed the E3 domain of human laminin ␣1 chain as a recombinant protein and made antibodies specific for human laminin ␣1 chain (anti-hLN-␣1G4/G5). We also made antibodies to the human laminin ␣5 chain purified from placenta. In the present report we show that hLN-␣1G4/G5 antibodies react with a 400-kDa laminin ␣1 chain and that 4C7 reacts with a 380-kDa laminin ␣5 chain. Immunohistochemistry with the hLN-␣1G4/G5 antibody and 4C7 revealed that the two antibodies stained human kidney, developing and dystrophic muscle in distinct patterns. Our data indicate that the previously reported expression patterns in developing, adult, and dystrophic human muscle tissues with 4C7 should be re-interpreted as an expression of laminin ␣5 chain. Our data are also consistent with earlier work in mouse, indicating that laminin ␣1 is largely an epithelial laminin chain not present in developing or dystrophic muscle fibers.
Cellular interactions with the extracellular matrix have been implied to be important for several stages of muscle development (1)(2)(3)(4). An intact linkage to the surrounding basement membrane has been demonstrated to be of importance also for muscle stability in the adult stage (5,6). During regeneration events following muscle damage, the basement membrane acts as a scaffold for the generation of new muscle fibers (7,8). It is thus important to understand the molecular composition of basement membranes in muscle. Laminin-2, with the chain composition ␣2, ␤1, ␥1, is present in the muscle lineage from early stages of development in the mouse (9,10) and is apparently the major laminin isoform in adult muscle basement membranes (11). The finding that genetic defects affecting laminin ␣2 can cause muscular dystrophy has highlighted the importance of laminin-2 for the structural integrity of muscle (12,13). Molecular compensation in certain forms of muscular dystrophies by increased expression of laminin chains may decrease the severity of the diseases. Some evidence for this has been obtained in immunohistochemistry studies with the antibody 4C7, which is one monoclonal antibody from a panel of antibodies raised against human placental laminins (14,15). These antibodies were generated prior to the current knowledge about the existence of multiple laminin isoforms. The 4C7 antibody does not react with the laminin ␣2 chain but has been considered to react with the human ␣1 chain (14,15). The antibody, commercially available under different names, has been widely used to detect human ␣1 chain (previously called A chain) both in muscle and non-muscle tissues (14, 16 -20). The 4C7 antigen has been detected in basement membranes of normal muscle, and in blood vessels in muscle tissue (21), and increased expression of it in muscular dystrophies has been documented in numerous reports (22)(23)(24). It thus seemed reasonable to suggest molecular compensation by ␣1 chain in muscular dystrophies (22)(23)(24), particularly since many reports convincingly have shown that laminin-1 (␣1, ␤1, ␥1) can stimulate proliferation, motility, and development of muscle cells in vitro (1,25,26). Nevertheless, we and others have failed to detect laminin ␣1 chain in developing mouse muscle tissue (9,27,28), and no increased expression of this chain was seen in dystrophic mouse muscle (28). Furthermore, comparing the staining pattern in non-muscle tissue of 4C7 in human, rat, and hamster with the pattern seen in mouse and rat with other antibodies, there is a discrepancy. In non-neural tissues of mouse and rat the ␣1 chain is largely confined to epithelial basement membranes (27,29,30), but the 4C7 antigen is widely distributed in developing and adult human, rat, and hamster tissues (15,19,20). The staining pattern of 4C7 in human tissues is also in disagreement with the distribution of the ␣1 mRNA in human tissues (31,32). Antibody 4C7 might thus detect some other ␣ laminin chain, but this proposal is speculative (33,34) and has not been rigorously tested. Currently, five different laminin ␣ chains have been described (35) and 4C7 could potentially detect any of these or might detect several ␣ chains. In a cell line, 4C7 immunoprecipitated a large chain in the 400-kDa range together with 200-kDa chains, but the nature of the 400-kDa chain was not studied (15).
To clarify the discrepancies in laminin ␣1 distribution in mouse and human tissues, we made antibodies to the recombinant E3 domain of human laminin ␣1 and compared the specificity of these antibodies with that of 4C7. Immunoprecipitation of laminins from cell lines producing varying amounts of either ␣1 or ␣5 mRNA allowed a precise distinction of antibody specificity. Furthermore, we compared the distribution of laminin ␣1 chain and the 4C7 antigen in developing human muscle and in dystrophic human muscle tissue. Since the distribution of the laminin ␣1 chain in mouse kidney has been well described, we also analyzed the expression patterns in human kidney.

MATERIALS AND METHODS
Recombinant Laminin Expression-A 1180-base pair-long fragment from the 3Ј-end of human laminin ␣1 chain (nucleotide residues 8140 -9320) corresponding to the E3 region (carboxyl-terminal globular domain G4-G5) was amplified by PCR 1 from a 4.5-kb laminin ␣1 cDNA sequence ((36), clone number 7 supplied by E. Engvall The Burnham Institute, La Jolla Cancer Research Center) using AmpliTaq® (Perkin-Elmer). The primers were modified to include restriction sites for NotI and NheI to facilitate cloning into the expression vector. Primer sequences were as follows: forward primer, 5Ј GCC CCG CTA GCT CCC GAT GCA GAG GAC AGC A 3Ј; reverse primer, TCA GTT GCG GCC GCT CAG GAC TCG GTC CCA GG. The obtained PCR product was ligated into a TA-vector (PCR II™, Invitrogen) for sequence confirmation. Sequencing was performed with a Pharmacia T7 Sequencing™ kit (Pharmacia Biotech Inc.). The sequenced PCR product was cleaved with NotI/NheI and inserted into the episomal pCEP-Pu vector (which is a modified pCEP4 (Invitrogen) vector, provided by E. Pöschl Institute of Experimental Medicine, Friedrich-Alexander-University, Erlangen, Germany). The insertions sites were sequenced prior to transfection. 10 6 human embryonic kidney cells 293 EBNA (Invitrogen, Catalog number R-620-07) were stably transfected with 15 g of hLN-␣1G4/G5 in pCEP-Pu using lipofectAMINE™ reagent (Life Technologies, Inc.), according the instructions from the manufacturer. Transfected cells were selected in 2 g/ml puromycin and 0.25 mg/ml G418 (Life Technologies, Inc.) and the medium from cells grown under serum-free conditions was analyzed for recombinant protein by SDS-PAGE. Purified protein was separated on a 10% SDS-PAGE under reducing conditions, visualized with Coomassie Brilliant Blue, excised, and digested "in-gel" with trypsin according to Ref. 37. Liberated peptides were further analyzed as described in Ref. 38. One peptide (SPQVQSFDFS) was analyzed and found to be identical with amino acids 3048 -3057 in Ref. 36.
Antibodies-For the generation of antibodies to human laminin ␣1, medium was collected from confluent 293 EBNA hLN-␣1G4/G5 cells under serum-free conditions and supplemented with 1 mM benzamidine, 1 mM EDTA, 1 mM N-ethylmaleimide. Collected medium was diluted 1:2 in water, passed over a 10-ml DEAE-Sepharose® Fast Flow (Pharmacia Biotech Inc.) column, serially connected to a 5-ml Hi trap Heparin-Sepharose® column (Pharmacia Biotech Inc.). The DEAE column was disconnected, and the heparin column was washed in 0.1 M NaCl, 20 mM Tris-HCl, pH 8.0, prior to eluting in 0.3 M NaCl in 20 mM Tris-HCl, pH 8.0. Peak fractions containing recombinant protein were concentrated on a second 1-ml Hi trap Heparin-Sepharose column, and the resulting peak fraction was used for immunizations of two rabbits, using 50-g injections intramuscularly at intervals of 3 weeks. For immunohistochemistry, the antibodies were affinity-purified on the recombinant protein as described in Ref. 39 prior to staining. The polyclonal antibody to human laminin ␣5 was generated as follows: an extract from human placenta was purified by affinity chromatography on a laminin ␤1 chain antibody (Ab 545) as described in Ref. 40. A major 380-kDa purified protein band on SDS-PAGE was cleaved with trypsin and microsequencing of resulting peptides revealed laminin ␣5 sequences (see "Results"). The original 380-kDa SDS-PAGE band was used to generate the rabbit polyclonal antibodies to laminin ␣5 chain. The monoclonal antibody recognizing laminin ␤1/␥1 chains (clone 4C12.8) was obtained from Immunotech. The polyclonal antibody to intact mouse laminin-1 was from Sigma (L9393). The 4C7 antibody (sold under the name mAb 1924) was from Chemicon. To visualize the proximal tubules a polyclonal antibody specific for a brush border antigen of proximal tubules was used (41).
Immunoprecipitation and Western Blotting-JAR cells (human choriocarcinoma cells ATCC No HTB-144), RD (rhabdomyosarcoma ATCC No CCL-136), WWCS-1 (Wilm's tumor cell line (42)), and G6 (cloned primary human fetal myoblasts (43)) were grown in Dulbecco's modified Eagle's medium under standard conditions. Cells were labeled over-night in in the presence of 25 Ci/ml [ 35 S]methionine/cysteine (pro-Mix 35 S cell labeling mix (Amersham Corp.)). Medium was collected from cells, centrifuged, and supplemented with protease inhibitors (1 mM benzamidine, 1 mM EDTA, 1 mM N-ethylmaleimide). The centrifuged medium was processed for immunoprecipitation as described (44). For Western blotting, conditioned medium was collected from JAR cells. Medium was passed over a Ricinus communis agglutinin I-agarose column (Vector Laboratories), washed extensively in phosphate-buffered saline, and eluted with 0.5 M D(ϩ)-galactose (Sigma). Eluted proteins were directly used for immunoprecipitation. Immunoprecipitated proteins were solubilized in SDS-PAGE sample buffer and resolved on a 5% SDS-PAGE gel under reducing conditions. Separated proteins were transferred to nitrocellulose membranes in a Trans-Blot cell (Bio-Rad). Membranes were incubated with primary antibody, washed in TBS ϩ 0.05% Tween 20, followed by peroxidase-coupled sheep antirabbit IgG (Amersham) and developed using the ECL system (Amersham).
Immunohistochemistry-Serial sections, 5-8-m thick, of muscle biopsies of boys referred for diagnostic purpose and shown to lack dystrophin, of muscle samples of human fetuses with a gestation age of 22 weeks, and of biopsies of human kidneys were cut in a Reichert Jung cryostat at Ϫ25°C. The sections were collected on individual slides as well as on the same slide (one sample of human muscular dystrophy, one of human fetal muscle, and one of human kidney) to allow a direct comparison of staining intensity in the three different types of samples. The staining procedure was carried out as described in Ref. 28.
Northern Blot Analysis-Total RNA was isolated using Qiagen RNeasy midi kit according to the manufacturer's instructions. Northern blotting was performed as described (28). For laminin ␣1, the 1180-base pair-long fragment used for recombinant laminin expression was used as a probe. For laminin ␣5 a reverse transcription PCR amplified mouse cDNA (nucleotides 290 -891) was obtained from newborn mouse kidney total RNA and used as a probe as described (30). In addition a 1.3-kb human EST clone (accession W67855) was obtained from the Integrated Molecular Analysis of Genomes and their Expression (I.M.A.G.E.) consortium (I.M.A.G.E. consortium Clone ID:342926, United Kingdom Human Genome Mapping Project Resource Center, Hinxton, Cambridge, United Kingdom) and was sequenced from the vector T7 site and in a span of 360 nucleotides found to show 73% identity to mouse laminin ␣5 chain (nucleotides 9874 -10233) (45) and to be identical with a partial cDNA sequence for human laminin ␣5 in nucleotides 1903-2262 (46). The opposite end of the EST clone was found to be identical to the untranslated end of the partial human laminin ␣5 cDNA sequence (nucleotides 2891-3125) (46). The 1.3-kb fragment was excised with NotI/EcoRI and used as a probe.

Generation of Polyclonal Antibodies to Human Laminin-1 E3
Region-To obtain reagents specific for human laminin ␣1 for immunohistochemistry on human tissues, we stably expressed cDNA coding for the E3 region (carboxyl-terminal globular domains G4-G5) of laminin ␣1 episomally in 293 EBNA cells. Recombinant protein (hLN-␣1G4/G5) displayed an estimated molecular mass of 45 kDa under nonreducing conditions, shifted mobility to 55 kDa upon reduction, and bound heparin-Sepharose (Fig. 1). Following purification on DEAE-and heparin-Sepharose the hLN-␣1G4/G5 (Fig. 1) was used as an immunogen to generate polyclonal antibodies. These antibodies are further characterized below. We also generated an antibody to human laminin ␣5 chain. The polyclonal rabbit antibody, anti-hLN␣5, was raised to a 380-kDa SDS-PAGE band purified from human placenta by immunoaffinity chromatography on anti-laminin ␤1 IgG (data not shown). Amino acid sequencing of tryptic peptides obtained from the 380-kDa band revealed two peptides, which were identified in the deduced amino acid sequence from a recently identified partial human laminin ␣5 cDNA sequence (46) (Table I).
Northern Blot Analysis of Laminins-To characterize the antibodies we had generated we tested a number of cell lines in Northern blotting for their expression of laminin ␣1 mRNA. As a comparison we also probed for laminin ␣5 mRNA. Laminin ␣1 mRNA was only detected in JAR cells, whereas laminin ␣5 mRNA was detected at moderate levels in JAR cells and RD cells and and at lower levels in WCCS-1 cells (Fig. 2A). The size of laminin ␣1 mRNA was estimated to approximately 10 kb, and the laminin ␣5 mRNA, as detected by both laminin ␣5 probes, was distinctly larger. The size was in agreement with the previously reported size of 11-12 kb in mouse (45).
The monoclonal antibody 4C7 has been suggested to recognize laminin ␣1 chain. However, when medium from metabolically labeled JAR cells was immunoprecipitated with anti-hLN-␣1G4/G5 and 4C7 in parallel, different laminin ␣ chain bands were obtained. Whereas anti-hLN-␣1G4/G5 precipitated the 400-kDa band, 4C7 precipitated a 380-kDa band (Fig. 2B). In mouse the laminin ␣5 chain has been reported to have an molecular mass of 380 kDa (47). From JAR cells anti-hLN␣5 precipitated a 380-kDa band (Fig. 2B). From RD cells lacking reactivity with anti-hLN-␣1G4/G5, antibodies to laminin ␤/␥ chains and anti-hLN␣5 still precipitated a laminin with a molecular mass of the ␣ chain of 380 kDa. A 380 kDa ␣ chain band was also precipitated from RD and G6 cells with 4C7 (data not shown). WWCS-1 cells, shown in Northern to lack laminin ␣1 mRNA and to express low levels of laminin ␣5 mRNA, only precipitated visible ␤/␥ chain complexes in immunoprecipitation under the conditions used.
Western blotting of JAR cell medium and proteins immuno-precipitated from this medium with antibodies to laminin ␤/␥ chains, and subsequently blotted with antibodies to hLN-␣1G4/G5 (Fig. 2C, lane a), revealed strong reactivity with the 400-kDa band. Anti-hLN␣5 reacted weakly with a 380-kDa band in the material precipitated by laminin ␤/␥ chains (lane b). As shown in C the material immunoprecipitated from JAR cells with the 4C7 antibody did not react with hLN-␣1G4/G5 antibodies (lane c), whereas Western blotting with anti-hLN␣5 resulted in reactivity with the 380-kDa band (lane d). We also performed silver staining to independently illustrate the size difference between the laminin ␣ chains precipitated by the two antibodies. Silver staining of proteins immunoprecipitated with anti-hLN-␣1G4/G5 revealed a distinct 400-kDa band together with 200 -220-kDa bands (lane e), whereas silver staining of 4C7 reactive material revealed the 380-kDa band in addition to 200 -220-kDa bands (lane g). The 200 -220-kDa bands were recognized in Western blotting by a polyclonal antibody to mouse laminin ␣1␤1␥1 chains (data not shown). Distribution of Laminin ␣1 and Laminin ␣5 in Fetal and Adult Human Tissues-When human adult kidney was stained with affinity-purified antibodies to human laminin ␣1 (anti-hLN-␣1G4/G5) and human laminin ␣5 (4C7), contrasting staining patterns were observed. Anti-hLN-␣1G4/G5 selectively stained a subset of proximal tubuli, whereas 4C7 stained proximal and distal tubuli, glomerular basement membranes, and blood vessels (Fig. 3, A-C). In agreement with previously reported data, 4C7 stained muscle fibers in addition to blood vessels in human fetal muscle (Fig. 4A). In biopsy material from a dystrophic DMD boy, 4C7 stained basement membranes of muscle fibers, blood vessels, and somewhat more intensely groups of small diameter regenerating muscle fibers (Fig. 4B). In contrast, the hLN-␣1G4/G5 antibody did not specifically stain either developing or dystrophic muscle tissue (Fig. 4, C  and D). DISCUSSION In some forms of muscular dystrophy the primary defect is a disturbed linkage between the muscle fiber and the basement membrane (5,6). This leads to muscle degeneration but also to a regeneration event where satellite cells are activated, replicate, and fuse to form new myofibers. During this process the basement membrane is used for migration and as a scaffold for the formation of new fibers (7,8). Little is known about the basement membrane components made during regeneration. Laminins (35) and collagen IV (48) exist as multiple genetically distinct isoforms, and agrin is subject to extensive alternative splicing (49), opening up a wide spectrum of possible structural variations in the basement membranes synthesized by regenerating muscle fibers. One possibility is that some forms of laminins are up-regulated during regeneration. In a study of DMD, it was found that the laminin recognized by the antibody 4C7 was expressed in regenerating areas (23). The 4C7 antigen was also found to be induced in laminin ␣2-deficient congenital muscular dystrophy (24). Since 4C7 has been reported to detect laminin ␣1 chain, the results have been interpreted as an up-regulation of ␣1 chain in these muscular dystrophies. Nevertheless, no clear up-regulation of laminin ␣1 chain was seen in a mouse model of muscular dystrophy (28). The discrepancy seems to be due to antibody specificity, since we demonstrate here that 4C7 detects human laminin ␣5 chain and not the ␣1 chain. This gradually became apparent during our efforts to study laminin ␣1 chain expression in human muscular dystrophies.
To study a possible up-regulation of laminin ␣1 chain in human muscle diseases, we raised an antibody against a recombinant fragment of human laminin ␣1 chain. A cDNA clone covering the most carboxyl-terminal globular domains (G4-G5)

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Human laminin-␣5 partial RQATGDYMGVSLR cDNA residue #46 ͉ ͉ ͉ ͉ ͉ ͉ ͉ ͉ ͉ ͉ ͉ ͉ Tryptic peptide QATGDYMGVSLR of the ␣1 chain was transfected into mammalian cells to produce recombinant protein hLN-␣1G4/G5. This was used as an immunogen to produce polyclonal antibodies. The antibodies against hLN-␣1G4/G5 did not react with human muscle tissues. Using several assays, we therefore tested whether 4C7 shows a reactivity similar to the hLN-␣1G4/G5 antibody. We made a polyclonal antibody against purified human laminin ␣5 chain for comparison. Medium from cell lines, which by Northern blotting could be shown to produce variable amounts of either ␣1 or ␣5 mRNA, were used for immunoprecipitation with the different antibodies. JAR cells, which produced both chains, proved particularly important for this analysis, and other cell lines served as excellent controls. From the medium of JAR cells, our hLN-␣1G4/G5 antibody and 4C7 antibody precipitated heterotrimers with different ␣ chains. Both antibodies precipitated two similar 200-kDa bands assumed to be ␤1 and ␥1 chain, based on reactivity in Western blotting with antibodies to mouse laminin-1 (recognizing human ␤1/␥1 chains in immunoblotting). Whereas precipitates obtained with antibody hLN-␣1G4/G5 contained a polypeptide with the expected molecular mass of 400 kDa, those of 4C7 contained a slightly smaller 380-kDa polypeptide. Recent studies in mouse have shown that the ␣5 chain is slightly smaller than ␣1 (47). Silver staining of immunoprecipitated material on gels also revealed that hLN-␣1G4/G5 antibody detected a larger protein than 4C7. It has been suggested recently that 4C7 might recognize the ␣1 chain together with other chains (45), but we found no evidence for reactivity of 4C7 with ␣1 chain in either assay. By immunoblotting, the 380-kDa polypeptide could be identified as laminin ␣5 chain. The used antibody to laminin ␣5 was raised against an excised 380-kDa SDS-PAGE band, which by peptide microsequencing was identified as human laminin ␣5. Moreover, in cell lines shown by Northern blotting to produce low amounts of ␣5 chain mRNA, we failed to immunoprecipitate any bands with 4C7 or the polyclonal antibody to laminin ␣5. Finally, we tested the different antibodies in immunofluorescence and found that antibody hLN-␣1G4/G5 and 4C7 gave the same expression pattern in human tissues as antibodies against mouse laminin ␣1 chain and ␣5 chain in mouse tissues. These results are in agreement with the mapping of all five laminin ␣ chains by Miner et al. (34), which concluded that ␣5 was the most widely expressed, and ␣1 was the most restricted. Based on these studies we conclude that our hLN-␣1G4/G5 antibody detects human ␣1 chain and may at present be the only antibody with a documented true specificity toward human ␣1 chain, which is useful in immunohistochemistry, Western blotting, and immunoprecipitations. In contrast, we show that 4C7 is a specific monoclonal antibody for laminin ␣5 chain. It is likely that the other monoclonal antibodies described by Engvall et al. (15) to detect larger 300 -400-kDa laminin chains also detect ␣5 chain, but this remains to be shown. The identification of laminin ␣5 chain as the sole antigen of 4C7 antibody clarifies several much debated issues concerning the distribution of laminin ␣ chains.
The implication of our current findings for previous data obtained in muscle is thus that human laminin ␣5 is expressed in developing muscle and that this "embryonic" muscle laminin isoform is re-expressed in the regenerating muscle basement membrane in dystrophic muscle. In contrast, laminin ␣1 cannot be detected in human fetal or regenerating muscle basement membranes, in accordance with previous data from mouse tissues (9,27,28). In dystrophin-deficient forms of muscular dystrophy, a recent therapeutic approach is to try and induce expression of the dystrophin homologue utrophin (50). A similar approach might be feasible in laminin-2-deficient congenital dystrophies, where a compensatory up-regulation of another laminin at an early stage of the disease might be beneficiary. Laminin-1 (chain composition ␣1, ␤1, ␥1) clearly stimulates myogenesis in vitro (1,25,26). In the light of the present findings it might nevertheless be more relevant to test whether laminin-10 (chain composition ␣5␤1␥1) can stimulate myogenesis in vitro. Moreover, it will be essential to determine whether the induction of laminin ␣5 reduces the severity of the disease in laminin-2-deficient congenital muscular dystrophy.
The current findings have broad implications also for several open issues concerning the nature of laminin isoforms in nonmuscle tissues. One of the few major controversies in the laminin field has been the seemingly simple issue of the distribution of the ␣1 chain. This seems to be largely resolved by the current results. The 4C7 antibody, commercially available under different names, has been much used to study the distribution of laminin ␣1 chain in human, hamster, rat, and guinea pig tissues. The 4C7 antibody described by Engvall et al. (15) should not be confused with a more recent, well described laminin antibody recognizing laminin-5 (␣3␤3␥2) also named 4C7 (51). A data base search revealed numerous publications that have used the first described 4C7 antibody to demonstrate a broad tissue distribution of the antigen. All these results are different to the findings of a somewhat more limited distribution of ␣1 chain in mouse and rat embryonic and adult tissues (27,29,30,52). Here we therefore studied the distribution of laminin ␣1 chain in adult human kidney with our antibody against recombinant human E3 fragment. It selectively stained the basement membranes of a subset of the proximal tubules. It is highly significant that no staining was seen in basement membranes of blood vessels, distal tubules, or the collecting ducts. This is in complete agreement with our previous findings in mouse (29) and rat kidney (30). These studies strongly suggest that the distribution of laminin ␣1 chain is similar in rodent and human adult kidneys. Based on our studies in muscle and kidney, we predict that the staining pattern in other tissues with anti-hLN-␣1G4/G5 or with other true ␣1 chain-specific antibodies will match the pattern of ␣1 immunoreactivity in mouse. This will be of some importance to study in the future, given the reports of the distribution of laminin ␣1 chain performed with antibody 4C7. Many of the studies performed with 4C7 antibody should be seen as valuable sources for descriptions of the distribution of ␣5 chain. It should be noted, however, that in the mouse there are two ␣5 mRNA transcripts, 9 and 12 kb, indicating the existence of two laminin ␣5 isoforms (34). Whether such isoforms exist in human is unclear, since we have so far noticed only one 12-kb mRNA in FIG. 4. Detection of laminin ␣1 and laminin ␣5 in human skeletal muscle. Immunofluorescence with 4C7 on cross-sections of human fetal muscle revealed the presence of laminin ␣5 in muscle fiber basement membranes and blood vessels (A), whereas a parallel section lacked detectable levels of human laminin ␣1 (C). In DMD patient sections 4C7 stained larger blood vessels, capillaries, and around small myofibers (B), whereas laminin ␣1 was not detected in a parallel section (D). Bar: 100 m. the cells we studied. We do not know whether 4C7 detects different splice variants of the ␣5 chains and whether they exist in human tissues. Although this particular detail is still unclear, it is evident that 4C7, that reacts at least with human, horse (own data), guinea pig, rat, and hamster tissues, will be useful for many studies of the major ␣5 chain isoform in many species.