Component Of Epithelial Basement Membranes Research Articles

Epithelial Basement Membrane Regeneration After PRK-Induced Epithelial-Stromal Injury in Rabbits: Fibrotic Versus Non-fibrotic Corneal Healing.

To study epithelial basement membrane (EBM) regeneration in non-fibrotic and fibrotic corneas after photorefractive keratectomy (PRK). Rabbits (120 total) had either epithelial scrape alone, -4.50 diopters (D) PRK, -9.00 D PRK, or no surgery. Immunohistochemistry was performed on cryofixed corneas at time points from unwounded to 8 weeks (four corneas at each time point in each group). Multiplex immunohistochemistry was performed for EBM components, including collagen type IV, laminin beta-3, laminin alpha-5, perlecan, and nidogen-1. Stromal cellular composition was studied by triplex immunohistochemistry for keratocan, vimentin, and alpha-smooth muscle actin (SMA). PRK-injured EBM significantly regenerated by 4 days after surgery. However, early TGF-beta-regulating perlecan incorporation into the nascent EBM declined 4 to 7 days after surgery in fibrotic corneas. Non-fibrotic corneas that had fully regenerated EBM (with all five components incorporated into the EBM) were transparent and had few SMA-positive myofibroblasts in the stroma. Conversely, corneas with defective nascent EBM that lacked perlecan developed many anterior stromal myofibroblasts and fibrosis at 3 to 4 weeks after surgery and had large amounts of collagen type IV in the nascent EBM and anterior stroma. Myofibroblasts synthesized perlecan but were incompetent to incorporate the heparin sulfate proteoglycan into the nascent EBM. Corneal transparency was restored over several months even in fibrotic corneas, and this was associated with a return of EBM perlecan, myofibroblast disappearance, and reabsorption of disordered extracellular matrix. Defective incorporation of perlecan into the regenerating EBM by subepithelial myofibroblasts, and likely their precursor cells, underlies the development and persistence of stromal fibrosis after PRK corneal injury. [J Refract Surg. 2022;38(1):50-60.].

Epithelial basement membrane of human decellularized cornea as a suitable substrate for differentiation of embryonic stem cells into corneal epithelial-like cells.

The ability to decellularize and recellularize the corneas deemed unsuitable for transplantation may increase the number of available grafts. Decellularized corneas (DCs) may provide a natural microenvironment for cell adhesion and differentiation. Despite this, no study to date has evaluated their efficacy as a substrate for the induction of stem cell differentiation into corneal cells. The present study aimed to compare the efficiency of NaCl and NaCl plus nucleases methods to decellularize whole human corneas, and to investigate the effect of epithelial basement membrane (EBM) of whole DCs on the ability of human embryonic stem cells (hESCs) to differentiate into corneal epithelial-like cells when cultured in animal serum-free differentiation medium. As laminin is the major component of EBM, we also investigated its effect on hESCs differentiation. The decellularization efficiency and integrity of the extracellular matrix (ECM) obtained were investigated by histology, electron microscopy, DNA quantification, immunofluorescence, and nuclear staining. The ability of hESCs to differentiate into corneal epithelial-like cells when seeded on the EBM of DCs or laminin-coated wells was evaluated by immunofluorescence and RT-qPCR analyses. NaCl treatment alone, without nucleases, was insufficient to remove cellular components, while NaCl plus nucleases treatment resulted in efficient decellularization and preservation of the ECM. Unlike cells induced to differentiate on laminin, hESCs differentiated on DCs expressed high levels of corneal epithelial-specific markers, keratin 3 and keratin 12. It was demonstrated for the first time that the decellularized matrices had a positive effect on the differentiation of hESCs towards corneal epithelial-like cells. Such a strategy supports the potential applications of human DCs and hESCs in corneal epithelium tissue engineering.

The corneal epithelial basement membrane: Master regulator of the stromal myofibroblast response to injury

AbstractThe corneal epithelial basement membrane (EBM) is positioned between basal epithelial cells and the stroma. This highly specialized extracellular matrix functions not only to anchor epithelial cells to the stroma but also during migration, differentiation, and maintenance of the differentiated epithelial phenotype. Basement membranes are composed of a diverse assemblage of extracellular molecules, some of which are likely specific to the cornea. In general, they are composed of four primary components—collagens (types IV and VII), laminins, heparan sulfate proteoglycans, and nidogens. Our recent studies have demonstrated defective regeneration of the EBM in corneas that develop haze after high correction photorefractive keratectomy (PRK) in rabbits or irregular PTK in mice. Likely this is attributable to myofibroblast generation from bone marrow‐derived and keratocyte‐derived precursor cells that persist in the subepithelial stroma and block keratocyte/corneal fibroblast contribution of EBM components. In turn, persistent EBM defects facilitate the development and persistence of myofibroblasts via augmented TGF beta and PDGF penetration into the anterior stroma. Similar mechanisms underlie corneal scaring after most injuries.

Ubiquitination of α5β1 Integrin Controls Fibroblast Migration through Lysosomal Degradation of Fibronectin-Integrin Complexes

Cell migration requires endocytosis and recycling of integrins, but it is not known whether degradation of these membrane proteins is involved. Here we demonstrate that in migrating cells, a fraction of the endocytosed fibronectin receptor, alpha 5 beta 1 integrin, is sorted into multivesicular endosomes together with fibronectin and degraded in lysosomes. This sorting requires fibronectin-induced ubiquitination of the alpha 5 subunit, and the activity of the endosomal sorting complex required for transport (ESCRT) machinery, which interacts with alpha 5 beta 1 integrin. Importantly, we demonstrate that both alpha 5 ubiquitination and ESCRT functions are required for proper migration of fibroblasts. We propose that ligand-mediated degradation of alpha 5 beta 1 integrin via the ESCRT pathway is required in order to prevent endosomal accumulation of ligand-bound integrins that might otherwise form nonproductive adhesion sites. Fibronectin and alpha 5 beta 1 integrin therefore are trafficked to lysosomes in a similar way to growth factors and their receptors.

The production of recombinant human laminin-332 in a Leishmania tarentolae expression system

Laminin (LM)-332 (α3β3γ2), a large heterotrimeric glycoprotein, is an essential component of epithelial basement membranes that promotes cell adhesion and migration. Here, we expressed human LM-332 using a novel protein expression system based on the trypanosomatid protozoan host Leishmania tarentolae. Plasmids containing cDNA encoding full-length β3 and γ2 subunits and truncated α3 subunit were sequentially introduced into L. tarentolae. A recombinant strain harboring the three subunits of human LM-332 efficiently formed heterotrimer and secreted it into the culture medium. Heterotrimeric recombinant LM-332 (rLM-332) could be purified from culture medium with one-step immuno-affinity chromatography. The eluted fraction contained all three subunits, as confirmed by immunoprecipitation and immunoblotting. The purified rLM-332 showed similar cell adhesion activity to rLM-332 purified from mammalian cells, indicating its proper folding and assembly. The obtained expression level was not high; however, we suggest that this expression system has the potential for mass production of LMs for tissue engineering.

Matrix Revolution: Molecular Mechanism for Inflammatory Corneal Neovascularization and Restoration of Corneal Avascularity by Epithelial stem Cell Transplantation

Corneal neovascularization (CNV) associated with severe limbal stem cell (LSC) deficiency remains a challenging ocular surface disease in that corneal inflammation may persist and progress, and the condition will not improve without lsc transplantation. A prominent feature after successful LSC transplantation is the suppression of corneal inflammation and cnv, which is generally attributed to the endogenous anti-angiogenic/anti-inflammatory factors secreted by corneal epithelial cells. In addition, corneal epithelial basement membrane (EBM) plays a unique role in the regulation of angiogenesis; several potent anti-angiogenic factors are derived from the matrix component of EBM, such as endostatin (from collagen xviii) and restin (from collagen xv). Also, angio-inhibitory thrombospondin and tissue inhibitor of metalloproteinase-3 are deposited in EBM. Moreover, the heparan sulphate proteoglycan in EBM can bind and sequester vegf and FGF-2 from activation. Recently, cultivated corneal epithelial transplantation (CCET) and cultivated oral mucosal epithelial transplantation (comet) have emerged as promising techniques for the treatment of LSC deficiency. When human limbo-corneal epithelial (HLE) cells are cultivated on cryopreserved amniotic membrane, production of endostatin, restin, and IL-1ra is enhanced. This highlights the significance of delicate epithelial-matrix interactions in the generation of anti-angiogenic/anti-inflammatory factors by HLE cells, and this may, in part, explain the rapid restoration of corneal avascularity following CCET. In addition, whether epithelial stem cells can persist after transplantation is the key for CCET and COMET. Emerging evidence of long-term survival of cultivated epithelial cells after transplantation suggest that epithelial stem cells can be isolated and cultivated in vitro, and can re-establish the epithelial phenotype in vivo. Taken together, the merits of enhanced anti-angiogenic activity and the preservation of corneal epithelial stem cells encourage further application of this tissue engineering technique for ocular surface reconstruction.

Integrin Trafficking Regulated by Rab21 Is Necessary for Cytokinesis

Adherent cells undergo remarkable changes in shape during cell division. However, the functional interplay between cell adhesion turnover and the mitotic machinery is poorly understood. The endo/exocytic trafficking of integrins is regulated by the small GTPase Rab21, which associates with several integrin alpha subunits. Here, we show that targeted trafficking of integrins to and from the cleavage furrow is required for successful cytokinesis, and that this is regulated by Rab21. Rab21 activity, integrin-Rab21 association, and integrin endocytosis are all necessary for normal cytokinesis, which becomes impaired when integrin-mediated adhesion at the cleavage furrow fails. We also describe a chromosomal deletion and loss of Rab21 gene expression in human cancer, which leads to the accumulation of multinucleate cells. Importantly, reintroduction of Rab21 rescued this phenotype. In conclusion, Rab21-regulated integrin trafficking is essential for normal cell division, and its defects may contribute to multinucleation and genomic instability, which are hallmarks of cancer.

Laminin-332 promotes the invasion of oesophageal squamous cell carcinoma via PI3K activation

Laminin-332 is major component of epithelial basement membrane, and has an important role in cell migration and tumour invasion. Recently, the phosphatidylinositol 3-kinase (PI3K) activation induced by laminin-332 during carcinogenesis or tumour invasion has been highlighted in skin squamous cell carcinoma. The expression of laminin-332 in 126 resected oesophageal squamous cell carcinoma (ESCC) specimens was immunohistochemically examined to determine its associations with the clinicopathological characteristics, and the effect of laminin-332 on the invasiveness and the PI3K activation was assessed by in vitro experiments using ESCC cell lines (ESCCs). Sections with immunostaining signals in >30% cancer cells, which were observed in 55 of 126 cases, were judged to be positive for laminin-332. The positivity was significantly correlated with pTNM stage and poor prognosis. Inactivation of the PI3K pathway by laminin-332 blocking antibody suppressed the invasiveness of TE8 cell line, which secreted laminin-332 at high level and had high PI3K activity. The addition of the purified laminin-332 activated the PI3K pathway and increased the invasiveness of TE11 cell line, which secreted laminin-332 at lower level and had low PI3K activity. The deactivation of PI3K pathway using the PI3K inhibitor decreased the invasiveness of ESCCs and the secretion of laminin-332 in vitro. The expression of laminin-332 was one of the prognostic factors of ESCC. Laminin-332 could provide the autocrine positive-feedback loop through PI3K activation, contributing the invasive ability. Therefore, the inhibitor of PI3K pathway might be useful as the anticancer therapies for ESCC.

Matrilysin 1 Influences Colon Carcinoma Cell Migration by Cleavage of the Laminin-5 β3 Chain

Matrilysin 1 [matrix metalloproteinase 7 (MMP7)] is one of the most important metalloproteinases expressed in human tissues. This enzyme is generally not expressed by normal differentiated epithelial colon cells, but has been shown to be up-regulated in human colon adenomas and adenocarcinomas. Little is known about the role of MMP7 in cell invasion and its involvement in proteolytic processes. By searching the ligands of MMP7 in the colonic carcinoma cells HT29, we identified laminin-5/laminin-332 (LN5) as a specific target for MMP7 enzymatic activity. LN5, composed of alpha3, beta3, and gamma2 chains, is an important component of epithelial basement membranes where it induces firm adhesion and hemidesmosome formation. In this study, we show that LN5 and MMP7 are coexpressed in HT29 cells as well as in HT29 xenograft tumors and human colorectal adenocarcinomas. We provide evidence that human LN5 is a ligand for MMP7 and that a specific cleavage occurs in its beta3 chain, giving rise to a carboxyl-terminal beta3 chain fragment of 90 kDa. We have identified the MMP7 cleavage site at position Ala(515)-Ile(516) in the beta3 chain. Videomicroscopic analysis of HT29 cells plated on LN5 substrates reveals that the MMP7-processed LN5 significantly enhances cell motility. Moreover, the delayed migration of HT29 cells obtained after specific inhibition of MMP7 reinforces the hypothesis supporting its involvement in cell migration. Altogether, our results show that MMP7 is likely to play a crucial role in the regulation of carcinoma cell migration by targeting specific proteolytic processing of the LN5 beta3 chain.

Isolation and Activity of Proteolytic Fragment of Laminin-5 α3 Chain

Laminin-5 (α3β3γ2) is an important component of epithelial basement membranes. The 190-kDa α3 chain undergoes extracellular cleavage within the carboxyl (C) terminus consisting of five globular domains (G1 to G5), producing the mature laminin-5 with the 160-kDa α3 chain. To understand the physiological meaning of this processing, we isolated the C-terminal fragments of the α3 chain from the conditioned media of two kinds of human cell lines. The amino-terminal sequence of the fragments suggested that the cleavage occurs at Gln1337-Asp1338 in the spacer region between the G3 and G4 domains. The G4–G5 fragment itself did not show significant activity, but it stimulated cell migration in the presence of a low concentration of the mature laminin-5, suggesting its regulatory role in cell migration.

Purification and characterization of the GDA-J/F3 antigen, a novel component of epithelial basement membranes

Purification and characterization of the GDA-J/F3 antigen, a novel component of epithelial basement membranes

Corneal epithelial basement membrane after experimental anterior stromal puncture in guinea pigs: Immunohistochemical study

We investigated alterations in the immunolocalization of the components of epithelial basement membrane (BM), type IV collagen, and laminin in guinea pigs subjected to anterior stromal puncture (ASP) of the cornea performed with a standardized needle. Localization of BM components beneath the corneal epithelium was indicated by laminin immunoreactivity. The BM was interrupted by needle punctures immediately after ASP. During healing, type IV collagen immunoreactivity was detected transiently in the BM of some of the ASP-treated corneas, but no reactivity was observed in normal epithelial BM. Development of type IV collagen immunoreactivity was probably caused by an alteration of the α-chains or by an unmasking of the antigenicity of this collagen type, which may be related to an increase in the adhesiveness of the epithelium following ASP.

Abnormalities of the Extracellular Matrix in Keratoconus Corneas

To study alterations of the extracellular matrix (ECM) and basement membrane (BM) components in human keratoconus corneas. Fifteen normal and 13 keratoconus corneas were characterized by immunofluorescence with antibodies to 23 ECM and BM components. Keratoconus staining patterns for posterior nonscarred regions and Descemet's membrane were normal. We focused on three areas of keratoconus corneas: (a) nonscarred anterior corneal regions, (b) scarred anterior and posterior corneal regions, and (c) gaps in Bowman's layer. In each of these areas, consistent ECM and BM changes could be found. Nonscarred regions showed decreased staining of the epithelial BM for entactin/nidogen, fibronectin, alpha 3-alpha 5 chains of type IV collagen, and chains of laminin-1. In contrast, scarred regions had greater than normal staining of the epithelial BM for these same components and also for laminin-5, perlecan, and type VII collagen. In the Bowman's layer gaps/breaks, focal fibrotic deposits containing type VIII collagen, fibrillin-1, tenascin-C, alpha 1-alpha 2 type IV collagen, and normal stromal ECM and epithelial BM components were seen. Fibrotic regions were largely restricted to areas where, because of the lack of Bowman's layer, the epithelium was in contact with the stroma. In a single keratoconus cornea, abnormalities in the ECM/BM patterns were not uniform. This may reflect locally increased protease activity (where few if any BM components are found) and ongoing wound healing (where more BM or ECM components or both are found).

Differential Modulation of Integrin Receptors and Extracellular Matrix Laminin by Transforming Growth Factor-β 1 in Rat Alveolar Epithelial Cells

The transforming growth factors-β (TGFs-β) family of genes plays important roles in cell growth and differentiation in many cell types. TGFβ modulates the synthesis and accumulation of extracellular matrix (ECM) components and the expression of cell surface receptors for ECM components. TGFβ is increased in alveolar lining fluid during inflammatory reactions of the lung and has been identified in alveolar epithelial cells of developing lungs and hyperplastic type II cells during repair. However, little is known about how TGFβ may regulate expression of extracellular matrix proteins and ECM receptors in lung alveolar epithelial cells. Laminin, a major glycoprotein component of epithelial basement membrane, is synthesized and secreted by alveolar epithelial cells. To study the effects of TGFβ on modulation of laminin and its integrin receptors α 6β 1 and α 3β 1 in lung alveolar epithelial cells, a rat alveolar type II cell-derived cell line, LM5, was incubated with TGFβ 1 (0-100 pg/ml) in serum-free medium for 0-16 h. We examined the expression of integrin subunits and laminin β2 chain (s-laminin) mRNAs and protein expression. By Northern blot analysis, TGFβ 1 induced dose-dependent increases in α 6 and β 1 mRNA levels. TGFβ 1 also increased the expression of laminin β2 chain mRNA at 12-16 h poststimulation. In contrast, TGFβ decreased α 3 mRNA expression. Immunoprecipitation studies of TGFβ 1-treated cells showed increased surface expression of both α 6 and β 1 protein while surface expression of the α 3 integrin subunit was decreased. The same treatment resulted in increased laminin protein expression. These data suggest that TGFβ 1 may regulate alveolar epithelial cell differentiation in part through its modulation of integrins and laminin chains.

Epithelial Basement Membrane in Alkali-Burned Corneas in Rats

We investigated the altered immunolocalization of the components of epithelial basement membrane (BM) in rats with alkali burns of the cornea. Type IV collagen, laminin, and fibronectin were immunohistochemically stained in the alkali-burned corneas after various intervals. Linear laminin immunoreactivity, which represented the localization of the BM, was interrupted, probably by epithelial enzymes, under the regenerating epithelium after 24 h. A normal configuration was restored on day 7, when the central corneal BM showed a type IV collagen immunoreactivity that was not detected in BM of normal cornea. The exact cause of the development of type IV collagen immunoreactivity in the healing BM was not determined. Laminin, type IV collagen, and fibronectin, which were not detected in normal stroma, were all detected in the healing corneal stroma with repopulated keratocytes. Moreover, the BM zone under the regenerating epithelium showed fibronectin immunoreactivity on days 5-14. Distribution of these components of BM seemed to be normal 2 months postburn. The alteration of BM components is thought to be an important marker of the healing process in corneas burned with alkali.

Epiligrin, a component of epithelial basement membranes, is an adhesive ligand for alpha 3 beta 1 positive T lymphocytes.

The cutaneous T cell lymphomas (CTCL), typified by mycosis fungoides, and several chronic T cell mediated dermatoses are characterized by the migration of T lymphocytes into the epidermis (epidermotropism). Alternatively, other types of cutaneous inflammation (malignant cutaneous B cell lymphoma, CBCL, or lymphocytoma cutis, non-malignant T or B cell type) do not show evidence of epidermotropism. This suggests that certain T lymphocyte subpopulations are able to interact with and penetrate the epidermal basement membrane. We show here that T lymphocytes derived from patients with CTCL (HUT 78 or HUT 102 cells), adhere to the detergent-insoluble extracellular matrix prepared from cultured basal keratinocytes (HFK ECM). HUT cell adhesion to HFK ECM was inhibitable with monoclonal antibodies (mAbs) directed to the alpha 3 (P1B5) or beta 1 (P4C10) integrin receptors, and could be up-regulated by an activating anti-beta 1 mAb (P4G11). An inhibitory mAb, P3H9-2, raised against keratinocytes identified epiligrin as the ligand for alpha 3 beta 1 positive T cells in HFK ECM. Interestingly, two lymphocyte populations could be clearly distinguished relative to expression of alpha 3 beta 1 by flow cytometry analysis. Lymphokine activated killer cells, alloreactive cytotoxic T cells and T cells derived from patients with CTCL expressed high levels of alpha 3 beta 1 (alpha 3 beta 1high). Non-adherent peripheral blood mononuclear cells, acute T or B lymphocytic leukemias, or non-cutaneous T or B lymphocyte cell lines expressed low levels of alpha 3 beta 1 (alpha 3 beta 1low). Resting PBL or alpha 3 beta 1low T or B cell lines did not adhere to HFK ECM or purified epiligrin. However, adhesion to epiligrin could be up-regulated by mAbs which activate the beta 1 subunit indicating that alpha 3 beta 1 activity is a function of expression and affinity. In skin derived from patients with graft-vs.-host (GVH) disease, experimentally induced delayed hypersensitivity reactions, and CTCL, the infiltrating T cells could be stained with mAbs to alpha 3 or beta 1 and were localized in close proximity to the epiligrin-containing basement membrane. Infiltrating lymphocytes in malignant cutaneous B disease (CBCL) did not express alpha 3 beta 1 by immunohistochemical techniques and did not associate with the epidermal basement membrane. The present findings clearly define a function for alpha 3 beta 1 in T cells and strongly suggest that alpha 3 beta 1 interaction with epiligrin may be involved in the pathogenesis of cutaneous inflammation.

Tissue-specific distribution of a novel component of epithelial basement membranes

Monoclonal antibodies against basement membrane (BM) were generated using the matrix deposited by cultured rabbit corneal epithelial cells as immunogen. BM antibodies were identified by immunofluorescent staining of frozen tissue sections and of extracellular matrix of living cultured cells. BM localization was confirmed by immunoelectron microscopy. Antibody AE26 immunoprecipitates a 140,000 M r component from radiolabeled corneal epithelial cells and recognizes this component plus a 95,000 M r band on Western blots. The antigen resists extraction by high and low salt and by nonionic detergents, but is solubilized in 4 M urea/1% mercaptoethanol. On isoelectric focusing and nonequilibrium pH gradient gels, AE26 antigen migrates to the acidic region (p I < 3). The molecule is destroyed by trypsin, but is insensitive to bacterial collagenase. In frozen tissue sections, AE26 stains only BM of stratified epithelia plus trachea, ureter, lung, and intestine, but no other epithelial or nonepithelial BM. AE26 antigen is detected on Western blots of cornea, skin, and lung extracts, but not liver, kidney, or muscle, indicating that this is not due to masking of the epitope. This tissue distribution is different from any previously described BM molecule. Although we have not ruled out the possibility that AE26 recognizes a modification or fragment of a known BM component (particularly entactin), the acidic p I, collagenase resistance, and unusual tissue specificity suggest that AE26 recognizes a new BM protein. The BM heterogeneity demonstrated by AE26 may play a structural role or provide positional signals to the overlying epithelium.

Distribution of fibronectin in the ectoderm of gastrulating chick embryos

A GLYCOPROTEIN known as large external transformation-sensitive (LETS) protein, cell surface protein (CSP) or fibronectin1–3 can be identified on the surface of various cell types1. This protein has a molecular weight of 210,000–250,000 and is closely related to the cold-insoluble globulin found in serum and to the factor needed for cell spreading in vitro4,5. In culture many cells secrete fibronectin into the growth medium and on to the substratum6–8; fibroblasts and other cells of mesodermal origin produce particularly large amounts. However, in the early mammalian embryo fibronectin is present in basement membranes before the mesoderm has formed, when the embryo consists only of ectoderm and endoderm9. Adult epithelia also produce fibronectin in vitro10, and it forms a component of epithelial basement membranes in vivo11–13. We have applied an immunofluorescent technique to whole mounts of early chick embryos to examine the distribution and possible morphogenetic function of fibronectin. We discuss our results here, and suggest that fibronectin is involved in morphogenetic movement during early development.

Differentiation in human amniotic fluid cell cultures: II: Secretion of an epithelial basement membrane glycoprotein.

Cells obtained by amniocentesis for prenatal diagnosis were grown in vitro and examined for the presence of a glycoprotein component epithelial basement membrane. Isolated colonies or clones of amniotic fluid-type cells secrete the glycoprotein, which was identified in association with the cells using indirect immunofluorescent antibody techniques. In addition, the glycoprotein was isolated from tissue culture medium and identified as a component of epithelial basement membranes by passive haemagglutination (PHA) and immunodiffusion assays. Fibroblast-type cells do not secrete the glycoprotein. These results correlate well with the synthesis of type IV collagen by amniotic fluid cells reported in the accompanying paper (Priest et al., 1977) and indicate that amniotic fluid cells are epithelial in origin.

Isolation and characterization of an epithelial basement membrane glycoprotein from murine kidney and further characterization of an epithelial basement membrane glycoprotein secreted by murine teratocarcinoma cells in vitro

A glycoprotein component of epithelial basement membranes (EBM) has been isolated from murine kidney homogenates by extraction with 0.05 M phosphate buffer, pH 7.2, precipitation with (NH 4) 2SO 4 and chromatography on controlled pore glass. Antiserum produced against this glycoprotein reacts specifically with the basement membranes of renal glomeruli and tubules. The EBM glycoprotein of renal origin is antigenically identical with a glycoprotein component of epithelial basement membrane secreted by a murine teratocarcinoma grown in vitro, and the amino acid composition of the two EBM glycoproteins is markedly similar. Both glycoproteins were isolated as high molecular weight aggregates. Disaggregation with sodium dodecyl sulfate and 2-mercaptoethanol resulted in release of monomers of 32 000 and 34 000 daltons for kidney EBM glycoprotein and teratocarcinoma EBM glycoprotein, respectively. The difference in molecular weight is apparently due to increased amounts of fucose, mannose, N- acetylglucosamine and sialic acid in the glycoprotein secreted by the teratocarcinoma. In addition, both EBM glycoproteins contain galactose, glucose and N- acetylgalactosamine .