Characterization of Laminin 12 (α2β1γ3)
Laminin 12 was extracted from human chorionic villi using EDTA and partially purified by a combination of DEAE-cellulose, Concanavalin A, Sephacryl S-500, and Mono-Q chromatography (see Materials and Methods). The final fraction of interest resulting from the above protocol contains multiple laminins. Laminin 12 was resolved from this mixture by SDS-PAGE (3–5% polyacrylamide) under nonreducing conditions. Six bands were resolved (Fig. , Unreduced). Only the bands at ~560 kD and at the top of the gel were reactive with a polyclonal anti-laminin antiserum (Sigma Chemical Co.
; not shown). Therefore, the resolved band at 560 kD was excised, reduced in 10% 2-mercaptoethanol SDS-PAGE sample buffer, and resolved by 5% SDS-PAGE. Three bands were observed with masses of ~205, 185, and 170 kD (Fig. , Reduced). The band at 185 kD reacted with a monoclonal antibody (clone 545; Marinkovich et al., 1992
) specific to the laminin β1 chain (Fig. , Western blot). Each of the three bands was digested with trypsin, the peptides were resolved by HPLC, and selected resolved peptides were sequenced. The sequences obtained are shown in Table . The 205-kD chain contained three peptides sequence identical to human laminin α2 (published residues 536, 70, 1367; Vuolteenaho et al., 1994
). On that basis, the band was identified as human laminin α2, despite our observation that the 205-kD band did not react with anti-α2 mAb (mAb 1922; Chemicon). The band at 185 kD produced two peptides identical to human β1, and was thereby confirmed as human β1.
Figure 1 Identification of laminin 12 isolated from human placenta. A partially purified preparation of laminins isolated from an EDTA extract of placenta was subjected to 3–5% SDS-PAGE (Unreduced). The gel band in the indicated position (bar) was excised (more ...)
Comparison of Peptide Sequences Obtained from Placental Laminin 12 205- and 185-kD Bands and Published Sequences of α2 and β1 Residues
In contrast to the easy identification of the other two bands, the band at 170 kD contained three sequences not contained within any known laminin chain. The NH2-terminal sequence of the 170-kD chain was determined, and it also was novel; i.e., nonidentical to known laminin sequences. As these four sequences from the 170-kD band were derived from an unknown laminin and we had identified the laminin α and β chains, we assumed these sequences were derived from a novel laminin γ chain that we call γ3.
The apparent molecular masses for the 205- and 185-kD bands are not consistent with the literature values published for the α2 and β1 chains, respectively. Thus, these bands are indicated in Fig. as α2t, β1t, and γ3t to indicate that they have been processed (truncated). Laminin 2 and laminin 4 were also present in these preparations; when characterized by similar procedures (not described here in detail) they showed molecular masses consistent with literature predictions, suggesting that our preparations were not extensively and nonspecifically degraded. Together these observations suggest that the truncations observed for the γ3-containing molecules may be physiologically relevant.
Characterization of the γ3 cDNA
The cDNA sequences of human γ1 and γ2 were used to probe the National Center for Biomedical Information (NCBI) expressed-sequence-tag database (dbEST), and a clone was identified that was homologous, but not identical, to γ1 and γ2. The sequence of this clone was used to design PCR primers for extensions at 3′ and 5′ ends (see Materials and Methods) using human placental cDNA, and additional sequence information was obtained by a combination of genomic DNA and placental cDNA sequencing. The resulting sequence is shown in Fig. . The deduced amino acid sequence contains regions with 100% identity to all three of the peptide sequences obtained from the 170-kD band (underlined in Fig. ). The nucleotide sequence reported in this paper has been submitted to GenBank/EMBL Data Bank with the accession number AF041835.
Figure 2 The complete amino acid sequence of human γ3 as predicted from the corresponding cDNA sequence. The amino acid positions are numbered in accordance to the homologous locations within laminin γ1 (Pikkarainen et al., 1988). A 19–amino (more ...)
The DNA sequence contains an open reading frame predicting 1620 amino acids, including a 19–amino acid-long putative signal peptide that closely meets the criteria described by Nielsen et al. (1997)
. The predicted cleavage site was confirmed by protein sequencing of the γ3 NH2
terminus; this sequence exactly matched the predicted amino acid sequence following the signal peptide. The overall sequence of γ3 is most similar to that of γ1, sharing 52% amino acid similarity with human γ1 (Pikkarainen et al., 1988
). In addition, the amino acid sequence predicted by the γ3 cDNA contains a domain distribution most like that of the γ1 chain. All six domains are represented.
Overall, the γ3 chain has 43.6% amino acid identity with the γ1 chain and 34% identity with the γ2 chain. The highest conservation is seen between domains γ1VI and γ3VI (Fig. ). Domains γ3V and III also show considerable similarity to domains γ1V and III and γ2V and III.
Figure 3 Comparison of the predicted domain structures and the percent amino acid sequence identity of γ3 with γ1 and with γ2. The γ3 cDNA predicts a full-length laminin chain where all the domains found in γ1 are represented. (more ...)
The predicted γ3 sequence contains nine potential glycosylation sites (Fig. , boxed), only two of which (Fig. , boxed and underlined) are conserved in both human and mouse γ1. As these conserved sites are contained within the globular domains IV and VI, it is likely that these sites are used physiologically. There is a single RGD sequence (boxed, hatched) within domain II, but this site is not conserved in either human or mouse γ1 and γ2 proteins. The sequence NVDPNAV (Fig. , double boxed) occurs within the fourth EGF-like repeat of domain III and is a homologue of the nidogen binding site (NIDPNAV) within the same domain of γ1. These sequences differ by only a single conservative amino acid substitution.
LAMC3 Maps to Chromosome 9q31-q34
The γ3 chromosomal location was determined by searching the NCBI Human Genomic Sequencing Index data base with the γ3 cDNA sequence. The sequence is identical to a database, Sequence Tagged Sites (clone WI-14302), that has been localized to chromosome 9q33-q34. A 1.2-kb γ3 cDNA probe within domains I and II of the predicted protein, the regions of least homology among the γ chains, was used to localize LAMC3 by fluorescent in situ hybridization (FISH) analysis (SeeDNA Biotech, Inc.). The results confirm the localization to chromosome 9q31-q34 (Fig. ).
Figure 4 Localization of LAMC3 to chromosome 9, band q31-q34 by FISH. The position of LAMC3 was probed using a 1.3-kb cDNA probe within predicted protein domains I and II of γ3. The FISH signal (A) was superimposed over the DAPI-banded chromosomes (B) (more ...)
Laminin γ3 Associates with α2β1 to Form Laminin 12 which, in Placenta, Is Lacking Part of the I/II Domains of All Three Chains
To determine the domains present within α2t, the 205-kD gel band, purified from placenta, was fragmented with trypsin and the resulting peptides were fractionated by HPLC; the masses of the eluted peptides were determined by mass spectroscopy. The ion chromatograms were then evaluated relative to the masses predicted from the published amino acid sequence for α2 in order to determine the NH2- and COOH-terminal peptides present within the digest. The results identified a number of tryptic peptides; among these, the peptide LVEHVPGQP(VR), beginning at residue 70 within domain VI, was the most NH2-terminal; the peptide GTTMTPPADLIEK, beginning at residue 1367 within domain III, was the most COOH-terminal. These results indicate that α2t is a fragment containing the short arm of the laminin α2 chain. This conclusion is consistent with the observation that the initial peptide sequence identified from α2t was within the short arm domains (above).
β1t and γ3t are also short arm fragments, as all the peptide sequences determined for both species are present within the short arm domains. However, the masses of α2t, β1t, and γ3t are greater than predicted for the short arms alone. In addition, α2t, β1t, and γ3t are not separable by gel electrophoresis without the reduction of disulfide bonds. Therefore, this truncated laminin 12 molecule is very likely to contain portions of domain II of all three chains, as the interchain disulfide bonds should lie between these domains. It is of interest to note that domain II of γ3 contains three cysteinyl residues whose bonding partners are not readily identified and are not present in domain II of other laminin chains. These three cysteinyl residues are conserved in mouse γ3 (Albus, A., and R.E. Burgeson, unpublished observation). Whether these cysteinyl residues could form intrachain, interchain, or intermolecular disulfide bonds that in some way contribute to the cleavage of γ3 chain-containing laminins is unknown.
Tissue Distribution of γ3 Expression by Northern Analysis
Tissue RNA blots and Master Blot dot blots (Clontech) were probed with a γ3 nucleotide probe (nt 1316 to 2277). A single major transcript of ~5 kb, consistent in size with other laminin γ chains, is present in several of the tissues examined (Fig. A). A small amount of a second larger transcript can also be detected. This larger transcript is most likely due to differences in polyadelylation or due to inefficient splicing. The γ3 chain RNA is abundant in spleen, testis, placenta, lung, and liver; lesser amounts are seen in kidney and ovary (Fig. A). The predominance of a single transcript allowed use of the RNA Master Blot (Clontech) to determine expression in a large number of other tissues. On this dot blot, tissue RNA concentrations have been normalized to housekeeping genes. The Master Blot (Fig. B) confirms the abundant presence of γ3 transcripts in placenta, adrenal gland, testis, lung, and fetal kidney, but also shows detectable levels of γ3 transcripts in numerous additional tissues, including brain and skeletal muscle.
Figure 5 Laminin γ3 chain is expressed widely in human tissues. The same cDNA probe described in Fig. was used to probe tissue blots (A) and a dot blot (B). Only a single major RNA species is detected on the tissue blots; strong signals (more ...)
Characterization of the Immunospecificity of Anti-Laminin γ3 (R16; R21)
A polyclonal antiserum, R16, was made in a rabbit to the γ3 chain excised from a reduced SDS-PAGE gel similar to that shown in Fig. . Another, R21, was made to recombinant γ3 protein (see Methods). The R16 antiserum recognizes the γ3 chain on immunoblots of placental extracts, but at very high antibody concentrations, it shows some reactivity with the β1 and γ1 chains as well. Thus, as a control, human neonatal foreskin was immunostained with anti-laminin γ1 (polyclonal anti-laminin 1; Sigma Chemical Co.
), anti-laminin γ2 (GB3, Verrando et al., 1987
), and with anti-laminin γ3 (R16). Crisp, brilliant fluorescence was observed along the dermal-epidermal junction, and around capillaries with the anti-γ1 antibodies (data not shown), and in the basement membrane at the dermal-epidermal junction with anti-γ2 (data not shown); in contrast, no signal above background was detected using the anti-γ3 reagent (R16) when it was applied at dilutions of 1:250 or more (data not shown). The antigen could not be unmasked by treatment of the cryosections with 2, 4, or 6 M urea, or with 2 M guanidinium-HCl (data not shown). As all known laminin chains have been detected in skin within either the epithelial basement membranes or the vascular basement membranes, these results indicate that the cross-reactivity detected by Western blot analyses using the polyclonal anti-γ3 (R16) antibody was either not apparent by immunohistochemistry, or was below detection at the antibody concentrations used. For the subsequent anatomical experiments (below), R16 was diluted 1:250 or greater to assure no cross-reactivity was occurring. R21, the affinity-purified antiserum to recombinant γ3, was also tested on sections of neonatal foreskin. As with R16, no immunoreactivity was seen (data not shown); thus we conclude that this antiserum has no cross-reactivity with other known γ chains. Neither R21 nor R16 antiserums label the blood vessel basement membranes (see below) consistent with a lack of cross-reactivity to other γ chains.
γ3-containing Laminins Are Localized to Peripheral Nerves and to Ciliated Epithelial Apical Surfaces
Unlike the lack of anti-laminin γ3 chain immunoreactivity seen in neonatal foreskin, laminin γ3 chain immunoreactivity was detected in human leg skin. As shown in Fig. A, and consistent with published results, laminin γ1 chain reactivity is seen at the dermal-epidermal junction and within the basement membranes of the vasculature, while laminin γ2 chain immunoreactivity is restricted to the dermal-epidermal junction (Fig. B). The laminin γ3 chain immunoreactivity is further restricted to distinct patches widely spaced along the dermal-epidermal junction (Fig. C). In experiments not shown, the immunoreactivity did not correlate positively or negatively with sites of cell proliferation, nor did it correlate with fixed positions relative to the rete ridges. However, there is a direct correlation of the laminin γ3 chain immunoreactivity (Fig. D) with sites where nerves cross the dermal-epidermal junction as detected by an antibody to the neuronal marker PGP9.5 (Fig. E), which reacts with ubiquitin COOH-terminal hydrolase (Day et al., 1990
). The results in skin suggest that γ3-containing laminins may be deposited into the dermal– epidermal junction by nerve or nerve associated cells, or that its expression by epithelial cells is induced by the adjacent nerve.
Figure 6 Laminin γ3 chain is expressed in restricted sites, correlated with nerve terminations, in human skin. Laminin γ1, γ2, and γ3 chains and nerve-specific ubiquitin COOH-terminal hydrolase (PGP 9.5) in human leg skin were (more ...)
Laminin γ3 is also expressed in the neural retina at the apical surface of the retina and in the outer synaptic layer (Libby, R.T., Y. Xu, E.P. Gibbons, M.-F. Champliaud, M. Koch, R.E. Burgeson, D.D. Hunter, and W.J. Brunken, manuscript submitted for publication); in the retina, the γ3 chain is coexpressed with the α4, α3, and β2 chains. Native γ3-containing laminins have not been isolated as yet from the retina; however, they have from another region of the central nervous system, the cerebellum, from which we have obtained two novel laminins, α3β2γ3 and α4β2γ3 (Champliaud, M.-F., unpublished observations). In addition, anatomical methods (immunohistochemistry and in situ hybridization), demonstrate the expression of γ3 in cerebellum and forebrain structures (Brunken, W.J., unpublished observations). It seems likely that γ3-containing laminins will be a general feature of the matrix in the CNS.
The Northern analysis indicated that the laminin γ3 chain was most strongly expressed in placenta, testis, lung, liver, spleen, and ovary. Therefore, we examined the localization of γ3 chains within testis, lung, and ovary. The reactivity within the epididymis and the fallopian tube were particularly striking. Thus, the distribution of γ3 in these tissues was extensively studied. In the female reproductive system, the oviduct was strongly reactive. Cryosections of the bovine (Fig. , A–F) or rat (Fig. , G–I) ampulla reacted for γ3 using R16 (Fig. , A, D, and G–I), or R21 (Fig. , B and E) showed brilliant immunoreactivity at the apical surfaces of the tubal mucosa. Double immunofluorescent studies performed with laminin γ3 and either laminin α2 (Fig. , A and D) or laminin α5 antibodies (Fig. E) demonstrated that both of these α chains are restricted to the basement membranes of the tubal epithelial and the subjacent endothelium whereas γ3 is expressed at the apical surface. The pre-immune serum from rabbit 21 (Fig. C) was negative, as was the reactivity of anti-thioredoxin antibodies purified from the R21 serum by immunoaffinity (Fig. F).
Figure 7 Laminin γ3 is expressed on the apical surface of the ciliated epithelium of the bovine and rat fallopian tube. Confocal laser microscope images of freshly frozen tissue sections of fallopian tube; in each image, five optical sections were (more ...)
The pattern of immunoreactivity for R16 in the rat oviduct was identical to that seen in bovine tissue (Fig. G). Higher magnification micrographs of the epithelial apical surface of the rat ampulla (Fig. , H and I) show the γ3 chain to be localized to the apical surface of the epithelial cells at the base of the cilia.
It should be noted that the labeling pattern of R21 differed somewhat from that of R16. In general, the pattern with R21 was somewhat punctate, showing large deposits of immunoreactivity at the apical surface, and increased cytoplasmic labeling of the tubal epithelium, whereas the R16 immunoreactivity was more restricted to the apical extracellular surface. These observations suggest that the R21 antiserum, made to recombinant domain I, may recognize the unfolded γ3 chain better than the R16 antiserum, which should recognize primarily short arm domains.
The male monkey reproductive tract was examined also. Like the fallopian tube, the epithelium in the epididymis is a single columnar epithelium (Fig. , A, H, and E). In situ hybridization performed on adjacent sections of the monkey epididymis (Fig. B; γ3) localized transcripts for the γ3 chain to the apical region of the epithelial cells (compare Fig. , A with B). R16 (data not shown) and R21 (Fig. , C and D, R21) sera gave similar patterns, reacting with both the basal and apical surfaces of the epithelial cells. The R21 antiserum reacted with apparently intracellular stores of γ3, as was seen in the bovine fallopian tube. The preimmune control serum from R21 showed only punctate autofluorescence (Fig. E, Pre).
Figure 8 Laminin γ3 is expressed on the surface of male reproductive epithelium. Light (A and B) and confocal (C–I) images of the epididymis from monkey (rhesus). In A, a section was fixed and stained with H&E for orientation; the (more ...)
Potential chains partners were explored by examination of the same tissue with antibodies specific for a variety of other laminin chains: α2 (Fig. F), α4 (G), β1 (H), and β2 (I). We used two monoclonal antibodies to test for the presence of β1 at the apical surface (clones 545; and C21) both gave the same pattern of immunolabeling; only the results with clone 545 are shown. As can be seen readily, α2 and β2 were restricted to the basal surface of the epithelial cells, while staining for α4 and β1 were also seen at the apical surface. Thus, in contrast to the results from placental extracts, α4 (and not α2) appears to be a candidate chain partner for γ3 in the epididymis. These observations suggest that a wide variety of γ3-containing laminins will be expressed in a tissue-specific pattern.
Expression of laminin γ3 chain was examined in the rat as well and the tissue distribution of γ3 in the rat epididymis was similar to that described for the monkey (data not shown); namely, γ3 immunoreactivity was localized to the apical surface of the epithelium. We also studied other regions of the rat reproductive system. Unlike laminin-1 immunoreactivity, which is localized to the basement membrane of the seminiferous tubules (Fig. A), γ3 immunoreactivity is not present within the basement membrane of the seminiferous tubules nor is it found around the interstitial cells (Fig. A, arrows; Fig. B, asterisk). Within the seminiferous tubules, only the occasional tubule reacted strongly with the laminin γ3 reactive serum (R16, Fig. B); it was our impression that those tubules identified by the antibody contained nearly mature spermatids. Further along the male reproductive system, in the ductus deferens, laminin-1 immunoreactivity (Fig. C; arrows mark the apical surface of the epithelium) was seen along the epithelial basement membrane, in the lamina propria and ensheathing the smooth muscle cells of the muscular layer. In contrast, γ3 immunoreactivity (R16) was found at the apical and basal surfaces of the epithelial cells, as well as intracellularly (Fig. D).
Figure 9 Laminin γ3 is expressed on the surface of rat ciliated epithelia in testis and lung. Various tissues from the rat male reproductive system (A–D) were incubated with either laminin-1 (α1/β1/γ1) antiserum (left (more ...)
The apical distribution of the γ3 chain is not confined to the reproductive system; in rat lung, the ciliated epithelial cells lining the bronchi were also strongly reactive with the anti-laminin γ3 antiserum, R16 (Fig. E). Again, the fluorescence was apparent along the apical surface, as determined by differential interference contrast microscopy (Fig. F). No γ3-immunoreactivity was seen in respiratory epithelium nor in the pulmonary capillary bed (not shown).