Mutations in the gamma 2 chain gene (LAMC2) of kalinin/laminin 5 in the junctional forms of epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is an autosomal recessive disorder characterized by blister formation within the dermal-epidermal basement membrane. Genes for the lamina lucida protein, kalinin/laminin 5, have been proposed as candidates for some forms of JEB, based on immunofluorescence analysis recognizing kalinin epitopes. We studied the cDNA of laminin gamma 2 chain for mutations in JEB using heteroduplex analysis. One patient showed a homozygous splice site mutation while another was heterozygous for a deletion-insertion, resulting in a premature termination codon in one allele. Our data implicate mutations in the laminin gamma 2 chain gene (LAMC2) in some forms of JEB.

Herlitz's junctional epidermolysis bullosa is linked to mutations in the gene (LAMC2) for the gamma 2 subunit of nicein/kalinin (LAMININ-5).

We have linked Herlitz's junctional epidermolysis bullosa (H-JEB) to the gene (LAMC2) encoding the gamma 2 subunit of nicein/kalinin, an isolaminin (laminin-5) expressed by basal keratinocytes. In four H-JEB kindreds, a maximum two-point lod score of 5.33 at theta = 0 was observed between a microsatellite near LAMC2 at 1q25-31 and the disease. In one family, a homozygous point mutation leading to a premature stop codon (CGA to TGA) was identified in exon 3 of the gene. The segregation of the mutated allele implicates the mutation in the pathology of the disorder and corroborates the linkage results.

Mutations in the laminin alpha 2-chain gene (LAMA2) cause merosin-deficient congenital muscular dystrophy.

Congenital muscular dystrophies (CMDs), are heterogeneous autosomal recessive disorders. Their severe manifestations consist of early hypotonia and weakness, markedly delayed motor milestones and contractures, often associated with joint deformities. Histological changes seen in muscle biopsies consist of large variations in muscle fibre size, a few necrotic and regenerating fibres and a marked increase in endomysial collagen tissue. Diagnosis is based on clinical features and on morphological changes. In several CMD cases, we have demonstrated an absence of one of the components of the extracellular matrix around muscle fibres, the merosin M chain, now referred to as the alpha 2 chain of laminin-2 (ref.3). We localized this CMD locus to chromosome 6q2 by homozygosity mapping and linkage analysis. The laminin alpha 2 chain gene (LAMA2) maps to the same region on chromosome 6q22-23 (ref. 5). We therefore investigated LAMA2 for the presence of disease-causing mutations in laminin alpha 2 chain-deficient CMD families and now report splice site and nonsense mutations in two families leading presumably to a truncated laminin alpha 2 protein.

The laminin family.

During the preceding year significant progress has been made in our understanding of laminin structure and expression. Highlights include the identification of new subunit chains and molecular isoforms as well as new information on tissue-specific gene expression, laminin self-assembly and interactions with other matrix components.

Role of the scavenger receptor MARCO in alveolar macrophage binding of unopsonized environmental particles.

Alveolar macrophages (AMs) avidly bind and ingest unopsonized environmental particles and bacteria through scavenger-type receptors (SRs). AMs from mice with a genetic deletion of the major macrophage SR (types AI and AII; SR-/-) showed no decrease in particle binding compared with SR+/+ mice, suggesting that other SRs are involved. To identify these receptors, we generated a monoclonal antibody (mAb), PAL-1, that inhibits hamster AM binding of unopsonized particles (TiO2, Fe2O3, and latex beads; 66 +/- 5, 77 +/- 2, and 85 +/- 2% inhibition, respectively, measured by flow cytometry). This antibody identifies a protein of approximately 70 kD on the AM surface (immunoprecipitation) that is expressed by AMs and other macrophages in situ. A cDNA clone encoding the mAb PAL-1-reactive protein isolated by means of COS cell expression was found to be 84 and 77% homologous to mouse and human scavenger receptor MARCO mRNA, respectively. Transfection of COS cells with MARCO cDNA conferred mAb-inhibitable TiO2 binding. Hamster MARCO also mediates AM binding of unopsonized bacteria (67 +/- 5 and 47 +/- 4% inhibition of Escherichia coli and Staphylococcus aureus binding by mAb PAL-1). A polyclonal antibody to human MARCO identified the expected approximately 70-kD band on Western blots of lysates of normal bronchoalveolar lavage (BAL) cells (>90% AMs) and showed strong immunolabeling of human AMs in BAL cytocentrifuge preparations and within lung tissue specimens. In normal mouse AMs, the anti-MARCO mAb ED31 also showed immunoreactivity and inhibited binding of unopsonized particles (e.g., TiO2 approximately 40%) and bacteria. The novel function of binding unopsonized environmental dusts and pathogens suggests an important role for MARCO in the lungs' response to inhaled particles.

Human basement membrane heparan sulfate proteoglycan core protein: a 467-kD protein containing multiple domains resembling elements of the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor.

The primary structure of the large human basement membrane heparan sulfate proteoglycan (HSPG) core protein was determined from cDNA clones. The cDNA sequence codes for a 467-kD protein with a 21-residue signal peptide. Analysis of the amino acid sequence showed that the protein consists of five domains. The amino-terminal domain I contains three putative heparan sulfate attachment sites; domain II has four LDL receptor-like repeats; domain III contains repeats similar to those in the short arms of laminin; domain IV has lg-like repeats resembling those in neural cell adhesion molecules; and domain V contains sequences resembling repeats in the G domain of the laminin A chain and repeats in the EGF. The domain structure of the human basement membrane HSPG core protein suggests that this mosaic protein has evolved through shuffling of at least four different functional elements previously identified in other proteins and through duplication of these elements to form the functional domains. Comparison of the human amino acid sequence with a partial amino acid sequence from the corresponding mouse protein (Noonan, D. M., E. A. Horigan, S. R. Ledbetter, G. Vogeli, M. Sasaki, Y. Yamada, and J. R. Hassell. 1988. J. Biol. Chem. 263:16379-16387) shows a major difference between the species in domain IV, which contains the Ig repeats: seven additional repeats are found in the human protein inserted in the middle of the second repeat in the mouse sequence. This suggests either alternative splicing or a very recent duplication event in evolution. The multidomain structure of the basement membrane HSPG implies a versatile role for this protein. The heparan sulfate chains presumably participate in the selective permeability of basement membranes and, additionally, the core protein may be involved in a number of biological functions such as cell binding, LDL-metabolism, basement membrane assembly, calcium binding, and growth- and neurite-promoting activities.

High expression of 92-kD type IV collagenase (gelatinase B) in the osteoclast lineage during mouse development.

cDNA clones for murine 92 kD type IV collagenase (gelatinase B) were generated for the determination of its primary structure and for analysis of temporal and spatial expression in vivo. The mouse enzyme has 72% sequence identity with the human counterpart, the major difference being the presence of a 16-residue segment absent from the human enzyme. In situ hybridization analyses of embryonic and postnatal mouse tissues revealed intense signals in cells of the osteoclast cell lineage. Clear expression above background was not observed in macrophages, polymorphonuclear leukocytes, monocytes, or epithelial cells which have been shown to express the gene in vitro in cell cultures. Expression of the gene was first observed at early stage of cartilage and tooth development at E13, where signals were seen transiently in surrounding mesenchymal cells. At later developmental stages and postnatally strong expression was seen in large cells at the surface of bones. These cells were presumably osteoclasts as their location correlated with that of TRAP positive cells. Signals above background were not observed in a number of other tissues studied. The results represent the first demonstration of a highly osteoclast specific extracellular proteinase. The results suggest that during normal development of embryonic organs the 92-kD type IV collagenase does not have a major role in basement membrane degradation, but is rather mainly used for the turnover of bone matrix, possibly as a gelatinase required for the removal of denatured collagen fragments (gelatin) generated by interstitial collagenase.

A truncated laminin chain homologous to the B2 chain: structure, spatial expression, and chromosomal assignment.

We describe the identification of a novel laminin chain. Overlapping clones were isolated from a human fibrosarcoma HT1080 cell cDNA library spanning a total of 5,200 bp. A second set of clones contained an alternative 3' end sequence giving a total of 4,316 bp. The longer sequence contained an open reading frame for a 1,193-residue-long polypeptide. The alternative sequence was shortened at the carboxyl-terminal end coding for a 1,111-residue-long polypeptide. The amino acid sequence contained 21 amino acids of a putative signal peptide and 1,172 residues or alternatively 1,090 residues of a sequence with five distinct domains homologous to domains I-V in laminin chains. Comparison of the amino acid sequences showed that the novel laminin chain is homologous to the laminin B2 chain. However, the structure of the novel laminin chain isolated here differs significantly from that of the B2 chain in that it has no domain VI and domains V, IV, and III are shorter, resulting in a truncated laminin chain. The alternative sequence had a shortened domain I/II. In accordance with the current nomenclature, the chain characterized here is termed B2t. Calculation of possible chain interactions of laminin chains with the B2t chain domain I/II indicated that the B2t chain can replace the B2 chain in some laminin molecules. The gene for the laminin B2t chain (LAMB2T) was localized to chromosome 1q25-q31 in close proximity to the laminin B2 chain gene. Northern analysis showed that the B2t chain is expressed in several human fetal tissues but differently from the laminin B1 and B2 chains. By in situ hybridization expression of the B2t chain was localized to specific epithelial cells in skin, lung, and kidney as opposed to a general epithelial and endothelial cell expression of the laminin B2 chain in the same tissues.

Human laminin M chain (merosin): complete primary structure, chromosomal assignment, and expression of the M and A chain in human fetal tissues.

The primary structure of the human laminin M chain was determined from cDNA clones isolated from human placental libraries. The clones covered a total of 6,942 bp, with 49-bp encoding a 5' end untranslated region and 6,893-bp coding for a translated sequence. The complete human laminin M chain contains a 22-residue signal peptide and 3,088 residues of the mature M chain. The M chain has a domain structure similar to that of the human and mouse A chains. The homology between the two human laminin heavy chains is highest in the short arm region and lowest in the long arm helical domain I + II. Northern blot analysis of human fetal tissues showed that the M chain was expressed in most tissues such as cardiac muscle, pancreas, lung, spleen, kidney, adrenal gland, skin, testis, meninges, choroid plexus, and some other regions of the brain, but not in liver, thymus, and bone. In situ hybridization localized the expression of the M chain gene to cells of mesenchymal origin. In contrast, expression of the A chain was observed only in kidney, testis, neuroretina and some region of brain as determined by Northern analyses. Epithelial and endothelial cells were negative for both M and A chain gene transcripts. The gene for the human M chain (LAMM) was localized to chromosome 6q22-->23.

Structure of human pro-matrix metalloproteinase-2: activation mechanism revealed.

Matrix metalloproteinases (MMPs) catalyze extracellular matrix degradation. Control of their activity is a promising target for therapy of diseases characterized by abnormal connective tissue turnover. MMPs are expressed as latent proenzymes that are activated by proteolytic cleavage that triggers a conformational change in the propeptide (cysteine switch). The structure of proMMP-2 reveals how the propeptide shields the catalytic cleft and that the cysteine switch may operate through cleavage of loops essential for propeptide stability.

Deletion of the paired alpha 5(IV) and alpha 6(IV) collagen genes in inherited smooth muscle tumors.

The gene encoding alpha 6(IV) collagen, COL4A6, was identified on the human X chromosome in a head-to-head arrangement and within 452 base pairs of the alpha 5(IV) collagen gene, COL4A5. In earlier studies, intragenic deletions of COL4A5 were detected in a subset of patients with Alport syndrome (AS), a hereditary defect of basement membranes. In some families, AS cosegregates with diffuse leiomyomatosis (DL), a benign smooth muscle tumor diathesis. Here it is shown that patients with AS-DL harbor deletions that disrupt both COL4A5 and COL4A6. Thus, type IV collagen may regulate smooth muscle differentiation and morphogenesis.

Identification of mutations in the COL4A5 collagen gene in Alport syndrome.

X-linked Alport syndrome is a hereditary glomerulonephritis in which progressive loss of kidney function is often accompanied by progressive loss of hearing. Ultrastructural defects in glomerular basement membranes (GBM) of Alport syndrome patients implicate an altered structural protein as the cause of nephritis. The product of COL4A5, the alpha 5(IV) collagen chain, is a specific component of GBM within the kidney, and the gene maps to the same X chromosomal region as does Alport syndrome. Three structural aberrations were found in COL4A5, in intragenic deletion, a Pst I site variant, and an uncharacterized abnormality, which appear to cause nephritis and deafness, with allele-specific severity, in three Alport syndrome kindreds in Utah.

Nephrin is involved in podocyte maturation but not survival during glomerular development.

Nephrin, a major component of the glomerular slit diaphragm (SD), is both a structural protein as well as a signaling molecule influencing foot process (FP) formation and maintenance of podocyte integrity. Analyses of near-term embryonic kidneys showed normal cellular viability and no apoptosis in glomeruli from nephrin knockout mice. Moreover, expression and location of other SD or glomerular basement membrane components were similar in wild-type and mutant mice as was the location and levels of most podocyte-specific proteins. Transcriptional profiling showed that the lack of nephrin had minor impact on the expression of genes for FPs and SD proteins. Claudin 3, a tight-junction protein normally absent in glomeruli, was upregulated threefold in the knockout mice, suggesting a role of nephrin in claudin 3 gene expression within the glomeruli. Our results suggest that nephrin is expressed late in the process of podocyte differentiation and is a locus for the formation of SD and FP maintenance and physical integrity in vivo. Nephrin does not seem to have a primary role in cell survival but has a small impact on gene regulation during glomerular development.

Congenital nephrotic syndromes.

Many acquired and familial renal diseases in man lead to kidney dysfunction and nephrotic syndrome. These diseases share a common pathological fate in the form of glomerular dysfunction and proteinuria. Classification of the disease is difficult because the onset of pathological appearance in congenital nephrotic syndrome (CNS) varies considerably. Recently, classification has been aided by applying molecular genetics to identify genes involved in the pathogenesis of proteinuria. Light has also been shed on the biology and mechanisms of glomerular filtration and the molecular pathogenesis of CNS.

Properly formed but improperly localized synaptic specializations in the absence of laminin alpha4.

Precise apposition of pre- to postsynaptic specializations is required for optimal function of chemical synapses, but little is known about how it is achieved. At the skeletal neuromuscular junction, active zones (transmitter release sites) in the nerve terminal lie directly opposite junctional folds in the postsynaptic membrane. Few active zones or junctional folds form in mice lacking the laminin beta2 chain, which is normally concentrated in the synaptic cleft. beta2 and the broadly expressed gamma1 chain form heterotrimers with alpha chains, three of which, alpha2, alpha4 and alpha5, are present in the synaptic cleft. Thus, alpha2beta2gamma1, alpha4beta2gamma1 and alpha5beta2gamma1 heterotrimers are all lost in beta2 mutants. In mice lacking laminin alpha4, active zones and junctional folds form in normal numbers, but are not precisely apposed to each other. Thus, formation and localization of synaptic specializations are regulated separately, and alpha4beta2gamma1 (called laminin-9) is critical in the latter process.

Deletions in the COL4A5 collagen gene in X-linked Alport syndrome. Characterization of the pathological transcripts in nonrenal cells and correlation with disease expression.

The type IV collagen alpha 5 chain (COL4A5) gene of 88 unrelated male patients with X-linked Alport syndrome was tested for major gene rearrangements by Southern blot analysis, using COL4A5 cDNA probes. 14 different deletions were detected, providing a 16% deletion rate in the COL4A5 gene in the patient population. The deletions are dispersed all over the gene with different sizes, ranging from 1 kb to the complete absence of the gene (> 250 kb) in one patient. In four patients with intragenic deletions, absence of the alpha 3 (IV) chain in the glomerular basement membrane was demonstrated by immunohistochemical studies. This finding supports the hypothesis that abnormalities in the alpha 5 (IV) chain may prevent normal incorporation of the alpha 3 (IV) chain into the glomerular basement membrane. Direct sequencing of cDNA amplified from lymphoblast mRNA of four patients with internal gene deletions, using appropriate combinations of primers amplifying across the predicted boundaries of the deletions, allowed us to determine the effect of the genomic rearrangements on the transcripts and, by inference, on the alpha 5 (IV) chain. Regardless of the extent of deletion and of the putative protein product, the 14 deletions occur in patients with juvenile-type Alport syndrome.

Laminin-5 as a marker of invasiveness in cervical lesions.

BACKGROUND: Treatment decisions for cervical cancer, a common disease worldwide, depend on demonstrating whether or not tumor invasion of the surrounding tissue has occurred. Invasion can be difficult to assess by standard histopathologic methods, especially when limited amounts of tissue are available. Several studies of a variety of cancers have reported increased expression of laminin-5-an important attachment protein for epithelial cells-in invasive carcinomas. This study was designed to investigate whether the presence of laminin-5 is related to the invasive capacity of cervical lesions. METHODS: We used immunohistochemical methods to stain archival, paraffin-embedded sections of cervical lesions with a polyclonal antibody specifically targeting the gamma2 chain of human laminin-5 protein. The study sample included 23 lesions of mild and moderate dysplasia (cervical intraepithelial neoplasia [CIN] 1 and 2, respectively), 32 lesions of severe dysplasia or carcinoma in situ (CIN 3), 15 lesions of microinvasive cancer, and 20 lesions of frankly invasive cancer. Cellular proliferative activity was also investigated by the use of monoclonal MIB-1 (directed against the antigen Ki-67) and anticyclin A antibodies. RESULTS: Invasiveness of cervical lesions was positively associated with immunohistochemical staining of the gamma2 chain of laminin-5 (two-sided P =.001). All CIN 1 and CIN 2 lesions-except one CIN 2 lesion later shown to be invasive cancer-and 21 CIN 3 lesions tested negative for the gamma2 chain of laminin-5. Eleven CIN 3 lesions and all invasive cancers tested positive for this protein. One lymph node metastasis and a pleural metastasis from one of the patients with invasive cancer showed strong immunohistochemical positivity. Proliferative activity increased with advancement of the lesion but was not confined to cells positive for the gamma2 chain of laminin-5. CONCLUSIONS: These data suggest that antibodies directed against the gamma2 chain of laminin-5 can identify cervical lesions with invasive capacity and thus may be useful as a sensitive marker of early invasion.

Localization of messenger RNA for Mr 72,000 and 92,000 type IV collagenases in human skin cancers by in situ hybridization.

We have examined the expression of 2 type IV collagen degrading enzymes (Mr 72,000 and 92,000 type IV collagenases) in human skin cancer by in situ hybridization. In all cases of infiltrating carcinomas of squamous cell (9 of 9) and basal cell (5 of 5) types, messenger RNA for the Mr 72,000 type IV collagenase was present in numerous fibroblasts. These were especially abundant in the stroma adjacent to the invasive tumor nodules. Malignant cells were negative for mRNA for the Mr 72,000 enzyme in all cases as were all other epithelial as well as endothelial cells. mRNA for the Mr 92,000 type IV collagenase was present in all 9 squamous cell and in 3 of the 5 basal cell carcinomas. In all these cases, a subpopulation of tissue macrophages was found to be positive, while malignant cells showed a signal for Mr 92,000 type IV collagenase in 6 of the squamous cell carcinomas but in none of the basal cell carcinomas. In all cases, the signal for this mRNA was confined to cells located at the tumoral/stromal interface or in the close vicinity of tumor nodules. No mRNA for any of the 2 collagenases was detected in 3 biopsies of normal skin. In vitro studies have indicated that collagenases are involved in the degradation of the extracellular matrix during cancer invasion. The present findings are consistent with such a role of the Mr 72,000 and 92,000 type IV collagenases in squamous and basal cell carcinomas in situ. The findings also demonstrate that degradative enzymes are not necessarily produced by the malignant cells themselves but may be generated by induction or recruitment of nonmalignant stromal cells.

Laminin-5 is a marker of invading cancer cells in some human carcinomas and is coexpressed with the receptor for urokinase plasminogen activator in budding cancer cells in colon adenocarcinomas.

Recombinant human gamma 2 chain of laminin-5 was expressed in Escherichia coli, and used to generate specific polyclonal antibodies which were used to study the distribution of the protein in human cancers. A total of 72 biopsies of human cancers were stained, including 23 cases of colon adenocarcinomas, 16 ductal breast carcinomas, 9 malignant melanomas, 14 squamous cell carcinomas of the skin and cervix, and 10 sarcomas. As a control for the specificity of the antibodies, we performed in situ hybridization on adjacent sections of a number of the cases, and in all of these cases the localization of the gamma 2 chain protein and mRNA was identical. We found gamma 2 chain immunoreactivity in cancer cells in all cases of colon adenocarcinomas and squamous cell carcinomas but not in any of the sarcomas, supporting the view that the laminin-5 protein is specific for cells of epithelial origin. Notably, in all of the cases of colon adenocarcinomas, the positive staining was invariably associated with budding cancer cells located at the tip of invading malignant epithelium, whereas the cancer cells deeper in the tumors were most often negative. The staining was cytoplasmic in all cases and only in one case did we see additional extracellular immunoreactivity, indicating that this laminin isoform in cancer tissue is not laid down in the extracellular matrix but probably exerts its function at the cell surface or in its immediate vicinity. Using in situ hybridization to analyze the coexpression of laminin-5 and components of the plasminogen activation system, we found that the histological distribution of laminin-5-positive budding cancer cells at the invasion front in colon adenocarcinomas was identical to that of the receptor for urokinase-type plasminogen activator. These findings suggest that laminin-5 is a marker of invading cancer cells in at least some human malignancies, and that it therefore might represent a valuable marker for the invasive potential of these cancers. The colocalization of laminin-5 and urokinase-type plasminogen activator receptor in a subset of cancer cells in colon cancer also suggests that a controlled up-regulation of a number of gene products is a characteristic of budding colon cancer cells, and that these gene products serve functions crucial for the invasive phenotype of these cancer cells.

Changes in intracellular enzymes of collagen biosynthesis during matrix-induced cartilage and bone development.

The activities of five intracellular enzymes of collagen biosynthesis were determined during cartilage and bone formation induced in rats by demineralized bone matrix. The five enzymes, prolyl 4-hydroxylase, prolyl 3-hydroxylase, lysyl hydroxylase, hydroxylysyl galactosyltransferase and galactosyl-hydroxylysyl glucosyltransferase, exhibited broadly parallel profiles; the activities rising steeply from day one to reach their highest values on day nine and decreasing gradually thereafter. The maximal enzyme activity correlated with the period of chondrogenesis and hypertrophic cartilage characterized by the synthesis of cartilage-specific type II collagen. Prolyl 4-hydroxylase was also studied in respect of its tissue distribution and cellular location using indirect immunofluorescence. The enzyme was mainly located in the mesenchymal cells on day three, in the chondrocytes and hypertrophic chondrocytes on days seven to nine, and in the osteoblasts on day eleven and thereafter.