Immunolocalization of several laminin chains in the normal human central and peripheral nervous system

Using specific monoclonal antibodies against different subunits of laminin, we studied the differential distribution pattern of several laminin chains in the central (CNS) and peripheral (PNS) nervous system. Laminin chains alpha 1, beta 1 and gamma 1, were found in the basement membrane (BM) of blood vessels in both CNS and PNS. In contrast, laminin alpha 2 though present in the BM of capillaries in the CNS, was completely absent from PNS capillaries. Laminins alpha 2, beta 1, gamma 1 could be detected in peripheral nerve, in the BM of Schwann cells, which did not contain Laminin alpha 1. The possible importance of laminin alpha 2 for myelination in the PNS as well as in the function of the blood-brain barrier in the CNS, and its potential relevance to the pathology of congenital muscular dystrophy associated with deficiency of this laminin chain, is discussed.     

Abnormal expression of laminin beta 1 chain in skeletal muscle of adult-onset limb-girdle muscular dystrophy

BACKGROUND: Laminin 2 is a major component of the basal lamina of skeletal muscle cells. It is a heterotrimer composed of 3 chains: merosin (laminin alpha 2 chain), beta 1, and gamma 1. Deficiency of merosin, with or without laminin beta 1 chain reduction, is associated with some forms of congenital muscular dystrophy. Deficient expression of laminin beta 1 chain is also associated with some cases of merosin-positive congenital muscular dystrophy. The expression of laminin 2 subunits has not been well studied in the skeletal muscle of limb-girdle muscular dystrophy (LGMD), nor has much attention been given to the significance of reduction of individual laminin 2 subunits, such as beta 1. OBJECTIVES: To examine the expression of laminin 2 subunits in skeletal muscle in patients with LGMD and to define the clinical features of patients with LGMD who have abnormal expression of laminin 2 subunits. METHODS: We studied muscle biopsy specimens from 18 patients with LGMD using immunofluorescence with antibodies against dystrophin C-terminus, beta-dystroglycan, alpha-sarcoglycan, gamma-sarcoglycan, and the laminin subunits merosin, beta 1, and gamma 1. Of the 18 biopsy specimens, 9 were available for electron microscopic examination of the muscle basement membrane. The clinical features associated with abnormal laminin beta 1 chain immunoreactivity were further described. RESULTS: Laminin beta 1 chain was either barely detectable or severely reduced in 3 cases of patients with LGMD in which the biopsy specimens showed normal staining with the other antibodies. Patients in all 3 cases had common clinical features consistent with a slowly progressive, adult-onset LGMD. Specimens from 2 of the 3 cases that were available for ultrastructural examination showed significant abnormalities of the muscle fiber basement membrane. CONCLUSIONS: Abnormal expression of laminin beta 1 chain without concomitant deficiency of alpha-sarcoglycan in skeletal muscle has not been previously described in LGMD. Reduced laminin beta 1 chain immunoreactivity may potentially serve as a marker for defining subsets of individuals with LGMD, in particular those with slowly progressive, adult-onset pelvifemoral presentation. The abnormality of muscle fiber basement membranes in specimens from cases that were available for ultrastructural study suggests that defects in the extracellular matrix may play a role in the pathogenesis of this subset of LGMD.     

Laminin localization in enterocytic basement membrane of rat small bowel grafts. A light and electron microscopic study

In vitro laminins stimulate numerous biological effects, such as cell migration, proliferation, attachment and differentiation. In vitro laminins influence immunocompetent cells and in vivo possibly play an important role in graft rejection. To establish how laminins could be involved in the regulation of acute rejection of small bowel allografts (with and without immunosuppression), we investigated laminin distribution in rat small bowel allografts four days after transplantation, i.e., before the onset of histological signs of rejection, using antibodies against alpha1, beta1, gamma1 chain of laminin-1. In immunosuppressed allografts, the ultrastructure of the enterocytic basement membrane appeared normal, but no laminin staining was seen in this membrane, although basement membranes of intramural blood vessels and muscle cells were normally stained. In non-operated immunosuppressed rats, laminin staining was clearly reduced in the enterocytic basement membrane, demonstrating that cyclosporin A is able to affect this membrane. Since only rats in which laminin is altered survive, this laminin alteration in the enterocytic basement membrane presumably plays an important role in overcoming the acute rejection.     

Palliative intubation of esophageal prosthesis in two patients with lung cancer

PURPOSE: Severe eye burns often result in extensive necrosis of the conjunctiva and episcleral tissue. Video fluorescein angiography was performed to reveal the perfusion of the anterior eye segment after severe eye burns. METHODS: A scanning laser ophthalmoscope was used for anterior segment fluorescein angiography in 12 patients (14 eyes) with severe burns grade III-IV and in 7 healthy volunteers. RESULTS: Necrotic tissues occurred as non perfused areas and remained dark throughout the whole angiogram. In general, the borders from healthy to necrotic conjunctival tissue were sharply demarcated. Thus, the extent of scleral and limbal ischemia could be determined exactly. Injured vessels showed hyperfluorescence with late leakage. Damage of the subconjunctival tissue appeared as a deep weak fluorescence in the early angiography and exhibited patchy leakage in the late angiogram. CONCLUSIONS: Anterior segment angiography provides a basis for deciding the extent of surgical debridement of necrotic tissue in the acute phase of the burn. The determination of the extent of limbal and scleral ischemia may give useful information for early plastic-reconstructive procedures.     

Specific attachment and migration of human astrocytoma cells on human but not murine laminin

Attachment sites and biological functions of laminin isolated from murine EHS sarcoma have been well studied. Recently several variants of laminin including human placental laminin have been shown to be distinct from EHS-laminin. This study was undertaken to determine attachment, proliferation, and migration phenomena of human astrocytoma cell lines to human and murine sarcoma EHS-laminin. Using short-term attachment assays human placental laminin was shown to be the better substrate for cell adhesion. EHS-laminin mediated approximately 30-50% of the effect observed on human laminin. The astrocytoma cells expressed beta 1, beta 3, and beta 4 subunit mRNA as determined by RT-PCR. Anti-beta 1 antibodies blocked adhesion to EHS-laminin, but antibodies against beta 1, beta 4, and alpha v subunits were all ineffective in blocking adhesion to human laminin. A migration assay showed that astrocytoma cells on human laminin dispersed from a central seeding area, while cells on EHS-laminin remained where they were seeded. The pattern of dispersion could not be accounted for by changes in growth rates of astrocytoma cells on the different proteins, since both cell lines grew equally well on the two laminins. We conclude that unique epitopes on human laminin are recognized by novel receptors on human astrocytoma cells which confer a migratory phenotype to the cells.     

Regulation of the beta 2 subunit chain of laminin in developing rabbit fetal lung tissue

Laminins are a family of basement membrane-associated heterotrimeric proteins that are important in mediating the growth, migration, and differentiation of a variety of cell types. The beta 2 subunit chain is a component of several laminin isoforms, e.g., laminin-3, laminin-4, laminin-7, and possibly other, as yet uncharacterized laminin isoforms. Utilizing monoclonal antibodies directed against the beta 2 subunit chain of laminin, we detected this protein in fetal, neonatal, and adult lung tissues. The relative amount of laminin beta 2 subunit chain in fetal lung tissue increased as gestation proceeded, reaching its peak around the time of alveolar type II cell differentiation in the rabbit. The laminin beta 2 subunit chain was localized in early gestational age rabbit fetal lung tissue primarily in basement membranes of prealveolar ducts, airways, and smooth muscle cells of airways and arterial blood vessels. A rabbit laminin beta 2 cDNA was generated using RT-PCR and utilized as a probe in northern blot analysis to characterize the levels of laminin beta 2 mRNA in developing rabbit lung tissue. Similar to the pattern of laminin beta 2 protein induction observed in fetal lung tissue, laminin beta 2 mRNA levels were maximal late in gestation. Utilizing a laminin beta 2 chain cRNA probe and in situ hybridization, we detected laminin beta 2 mRNA in the epithelial cells of prealveolar ducts, the alveolar wall, and airways, as well as in connective tissue cells, and the smooth muscle cells of airways and blood vessels in fetal and adult lung tissues. In addition, using an in vitro explant model, we determined that alveolar type II cells are capable of synthesizing laminin beta 2 subunit mRNA and depositing this laminin subunit chain in the basement membrane beneath type II cells. The results of this study are suggestive that the laminin beta 2 chain may be involved in alveolar epithelial cell differentiation.     

Clotrimazole, an imidazole antimycotic, is a potent inhibitor of angiogenesis

PURPOSE: To evaluate the roles of fibroblast proteins in the remodeling of the subconjunctival connective tissue, we immunohistochemically assessed the expression of matrix metalloproteinases (MMP)-1 and -2, and the tissue inhibitors of matrix metalloproteinases (TIMP)-1 and -2 in cultured human subconjunctival fibroblasts and in normal and healing human subconjunctival connective tissue. METHODS: Cultured fibroblasts derived from human subconjunctival connective tissue and surgical specimens of normal and healing conjunctiva were immunostained with monoclonal antibodies directed against human MMPs and TIMPs and examined by light and electron microscopy. RESULTS: In the cultured fibroblasts, MMP-1 and TIMP-1 antibodies stained the cytoplasm in a fine granular pattern, suggesting localization of those proteins in the endoplasmic reticulum (ER) and Golgi apparatus. Antibodies to MMP-2 and TIMP-2 reacted with fibroblast cytoplasm in a granular pattern. Electron microscopy of those fibroblasts revealed MMP-1 and TIMP-1 immunoreactivity in the ER cisternae or on the membrane of the ER. In surgical samples, MMP-1 and TIMP-1 were immunohistochemically detected in healing subconjunctival tissue, but not in conjunctival epithelium or normal subconjunctival tissue. CONCLUSIONS: MMPs and TIMPs may be involved in remodeling of subconjunctival connective tissue and in fibroblast population after surgical interventions. These proteins may play a crucial role in the post-operative fibrotic process occurring during scar formation in subconjunctival tissue.     

Mac-2 binding protein is a cell-adhesive protein of the extracellular matrix which self-assembles into ring-like structures and binds beta1 integrins, collagens and fibronectin

Human Mac-2 binding protein (M2BP) was prepared in recombinant form from the culture medium of 293 kidney cells and consisted of a 92 kDa subunit. The protein was obtained in a native state as indicated by CD spectroscopy, demonstrating alpha-helical and beta-type structure, and by protease resistance and immunological analysis. It was highly modified by N- and O-glycosylation but not by glycosaminoglycans. Ultracentrifugation showed non-covalent association into oligomers with molar masses of 1000-1500 kDa. Electron microscopy showed ring-like shapes with diameters of 30-40 nm. M2BP bound in solid-phase assays to collagens IV, V and VI, fibronectin and nidogen, but not to fibrillar collagens I and III or other basement membrane proteins. The protein also mediated adhesion of cell lines at comparable strength with laminin. Adhesion to M2BP was inhibited by antibodies to integrin beta1 subunits but not to alpha2 and alpha6 subunits, RGD peptide or lactose. This distinguishes cell adhesion of M2BP from that of laminin and excludes involvement of lactose-binding galectin-3. Immunological assays demonstrated variable secretion by cultured human cells of M2BP, which was detected in the extracellular matrix of several mouse tissues.     

Multi-component T1 relaxation and magnetisation transfer in peripheral nerve

Corneas of diabetic patients have abnormal healing and epithelial adhesion, which may be due to alterations of the corneal extracellular matrix (ECM) and basement membrane (BM). To identify such alterations, various ECM and BM components and integrin receptors were studied by immunofluorescence on sections of normal and diabetic human corneas. Age-matched corneas from 15 normal subjects, 10 diabetics without diabetic retinopathy (DR), and 12 diabetics with DR were used. In DR corneas, the composition of the central epithelial BM was markedly altered, compared to normal or non-DR diabetic corneas. In most cases the staining for entactin/nidogen and for chains of laminin-1 (alpha1beta1gamma1) and laminin-10 (alpha5beta1gamma1 was very weak, discontinuous, or absent over large areas. Other BM components displayed less frequent changes. The staining for alpha3beta1 (VLA-3) laminin binding integrin was also weak and discontinuous in DR corneal epithelium. Components of stromal ECM remained unchanged even in DR corneas. Therefore, distinct changes were identified in the composition of the epithelial BM in DR corneas. They may be due to increased degradation or decreased synthesis of BM components and related integrins. These alterations may directly contribute to the epithelial adhesion and wound healing abnormalities found in diabetic corneas.     

Developmental expression of laminin beta 2 in rat retina. Further support for a role in rod morphogenesis

PURPOSE: The authors previously hypothesized that laminin beta 2 (S-laminin) plays a role in directing photoreceptor development. The aim of this study was to examine the temporal and spatial expression pattern of beta 2 laminins in rat retina to test this hypothesis. METHODS: Retinas from Sprague-Dawley rats were harvested on embryonic days (E) 14, 16, and 21, as well as on postnatal days (P) 2, 5, and 10. Cryostat sections were probed with antibodies directed against beta 2 laminin, laminin-1 (alpha 1-beta 1-gamma 1), and von Willebrand factor. RESULTS: At the onset of rod photoreceptor birth (E14), laminin beta 2 surrounds the cells of the retinal pigmented epithelium (RPE) and is present on the apical surface of the retinal neuroepithelium. At E16, laminin beta 2 persists on the apical surface of the neuroepithelium and the subjacent apical surface of the RPE. At birth, laminin beta 2 fills the matrix between the juxtaposed surfaces of the RPE and neuroepithelium; moreover, laminin beta 2 immunoreactivity penetrates the neural retina. Throughout postnatal development, laminin beta 2 immunoreactivity surrounds maturing inner and outer segments. Laminin beta 2 also is found in association with blood vessels in the neural retina itself, as well as with choroidal blood vessels; in both places, it is co-localized with an endothelial marker, von Willebrand factor, and laminin-1. CONCLUSIONS: The spatial and temporal expression of laminin beta 2 is consistent with its hypothesized role in rod development. Laminin beta 2 is in a unique position to interact with mitotically active cells (in early retinal development), uncommitted progenitors (in late embryonic development), developing rods (in early postnatal development), and mature outer segments (throughout adulthood). Together with our earlier functional data, these data support our hypothesis that this molecule is an important component of the interphotoreceptor matrix.     

Importance of nidogen binding to laminin gamma1 for branching epithelial morphogenesis of the submandibular gland

Epithelial-mesenchymal interactions are major driving forces for the development of most solid organs. The importance of these interactions was first shown for the embryonic submandibular gland more than 40 years ago. We here present evidence that interactions between two basement membrane components, nidogen (entactin) and laminin gamma1 chain, could be important for epithelial-mesenchymal interactions in this gland. Nidogen mRNA was detected by in situ hybridization in the mesenchyme, and yet the protein was detected in epithelial and endothelial basement membranes. The role of nidogen-laminin interactions for epithelial morphogenesis was studied by applying antibodies to submandibular gland organ cultures. Antibodies reacting strongly with the nidogen-binding site of laminin gamma1 chain drastically perturbed branching epithelial morphogenesis. Electron microscopy of the epithelial-mesenchymal interface showed that blocking antibodies disrupted the formation of the basement membrane. Epidermal growth factor was shown to increase the expression of nidogen in mesenchyme, and could counteract the effect of the blocking antibodies. We suggest that nidogen could be an important mesenchymal factor for submandibular gland development.     

Immunohistochemical localization of extracellular matrix proteins in luteal phase endometrium of fertile and infertile patients

The lack of expression of certain components involved in cell adhesion and migration is believed to contribute to endometrial dysfunction and implantation failure. The purpose of this study was to investigate whether luteal phase endometrium in women with unexplained infertility differs, with respect to specific extracellular matrix (ECM) proteins, from endometrium of normal fertile women. A panel of monoclonal antibodies to collagen type IV, fibronectin and laminin was used to characterize the localization of ECM components in the different endometrial compartments. Precisely timed endometrial biopsies obtained at 4, 7, 10 and 13 days following the luteinizing hormone surge were obtained from 22 normal fertile women (group 1) and 24 women suffering from unexplained infertility (group 2). Paraffin-embedded sections were labelled using the streptavidin-biotin alkaline phosphatase technique. In group 1, collagen type IV, fibronectin and laminin were absent from the luminal epithelium but present in stromal cells and the basement membrane of glands and blood vessels. In group 2, these components were absent from all endometrial regions using equivalent titres of antibody to those used in group 1. This suggests that the endometrium of women with unexplained infertility demonstrates defects in the distribution of certain ECM glycoproteins. A possible consequence of this defect may be implantation failure.     

Breast cancer associated mucin: a review

Rabbit polyclonal antibody against mouse EHS laminin was used to investigate the distribution and composition of laminin in the rat first molar tooth germ. Immunohistochemical analysis showed that laminin is expressed in the inner and outer epithelia of the enamel organ and in small blood vessels in the dental papilla and strellate reticulum. Immunoblots revealed that tooth germ laminin differs from EHS laminin. Tooth germ laminin contains beta chains, while the alpha 1 chain is substituted by a 300-kDa chain. Two-dimensional electrophoresis analysis of tooth germ extract showed that beta chains appeared as four spots with approximate pI values of 6.6, 7.5, 7.8 and 8.5. These results indicate that more than-one type of laminin isoform is present in the first molar tooth germ. Additionally, we have shown that despite the early degradation of tooth germ basement membrane, the laminin molecule is still intact at the time of birth.     

Expression of laminin alpha 1, alpha 5 and beta 2 chains during embryogenesis of the kidney and vasculature

Laminins, found predominantly in basement membranes, are large glycoproteins consisting of different subsets of alpha, beta and gamma chain subunits. To resolve conflicting data in the literature concerning coexpression of alpha 1 and beta 2 chains, expression of alpha 1 chain was studied with two different antisera against the E3 fragment of laminin alpha 1 chain. Expression of the alpha 1 chain was seen in several types of epithelial basement membranes throughout development, but its expression in rat glomerular basement membranes and some other types of epithelial basement membranes occurred only during early stages of development. By contrast, beta 2 chains were detected by immunofluorescence only during advanced stages of glomerulogenesis and vascular development. By Northern and Western blots, beta 2 chains were detected somewhat earlier, but in situ hybridization revealed that beta 2 chain was also confined to vasculature during the earlier stages. It thus seems that, in the tissues studied here, the expression of alpha 1 and beta 2 chains was mutually exclusive. To explore whether the newly described alpha 5 chain is expressed in locations lacking alpha 1 chain, expression of alpha 5 chain was studied by Northern blots and in situ hybridization. The alpha 5 chain was not uniformly expressed in all embryonic epithelial cell types but was present mainly in epithelial sheets which produce very little alpha 1 chain. There also appeared to be a developmental trend, with alpha 1 chain appearing early and alpha 5 later, in maturing epithelial sheets. The alpha 5 chain could be a major alpha chain of the adult glomerular basement membrane.     

Iron deposition at mineralization fronts and osteoid formation following chronic cadmium exposure in ovariectomized rats

Integrins are heterodimeric glycoproteins that have been found to undergo dynamic temporal and spatial changes in distribution in the endometrium during the menstrual cycle in women. Likewise the extracellular matrix (ECM) ligands for these receptors are likely to play a role in the establishment of a receptive endometrium. To develop primate models to study the role of these molecules in the cascade of molecular events leading to implantation, integrin expression and associated changes in ECM were investigated during the menstrual cycle and in early pregnancy in the baboon. Antibodies specific for the integrins (alpha(1-6) and alpha(v); beta1, beta3, and beta4) and ECM (laminin, collagen IV, fibronectin) were utilized. In addition, cytokeratin and alpha-smooth muscle actin were used as epithelial, stromal, and smooth muscle cell markers, respectively. Endometrium was obtained in duplicate or triplicate during the menstrual cycle and early pregnancy. Changes observed during the natural menstrual cycle were confirmed using ovariectomized, steroid-treated animals. Constitutively expressed integrins on the endometrial epithelium included the collagen/laminin receptors: alpha2, alpha3, alpha6, and beta4. The pattern of expression correlated well with the distribution of ECM in this tissue. Collagen IV was confined to the basement membrane of glandular epithelium and blood vessels. Laminin immunostaining was found in the basement membrane, mostly in the stroma of the basal region, in the glandular endometrium and vasculature. Fibronectin was present throughout the stroma but not in the basement membrane. The collagen receptor alpha1 beta1 and fibronectin receptor alpha4 beta1 appeared in the glandular epithelium in the luteal phase. As in the human, alpha1 and alpha4 disappeared from the glandular epithelium with the establishment of pregnancy. In contrast, the alpha4 beta3 vitronectin receptor appeared in the glandular epithelium only in pregnancy or following long-term steroid treatment with estrogen and progesterone but not during the time of uterine receptivity associated with the initial period of embryo attachment. Osteopontin, an ECM ligand for alpha(v) beta3, was coexpressed with this integrin in invading cytotrophoblasts, glandular epithelium, and decidualizing stromal cells. Decidualization in the baboon was associated with changes in integrin expression similar to those found in humans: there was an increase in alpha1, alpha3, alpha6, beta1, and alpha(v) beta3 in the decidualized stromal cells. Laminin and collagen IV expression also increased at the implantation site and throughout the endometrium. In contrast, fibronectin expression was most evident at the implantation site and corresponded to alpha5 expression on the invading cytotrophoblasts. In summary, marked similarities were found in the expression of ECM and the integrin receptors between the baboon and the human endometrium throughout the menstrual cycle and in pregnancy. Cycle-specific integrins, alpha1, and alpha4, were present on epithelial cells during the secretory phase. Delayed expression of alpha(v) beta3 in baboon endometrial glands correlated closely with the time of enhanced glandular secretory activity in this primate. The baboon appears to be an excellent model for the investigation of the role of integrins and ECM leading to successful implantation.     

The effects on inhibition of corneal neovascularization after human amniotic membrane transplantation in severely damaged rabbit corneas

Human amniotic membrane isolated from the placenta contained basement membrane components such as type IV collagen, laminin, and 6 and 4 integrins, all of which remained detectable while preserved in glycerin for one week. One month after the n-heptanol removal of the total corneal epithelium and the limbal lamellar keratectomy, all rabbit eyes carried features of limbal deficiency, including conjunctival epithelial ingrowth, vascularization and chronic inflammation. Ten control eyes then received a total keratectomy, and 13 experimental eyes received an additional amniotic membrane transplantation. Three-month follow-ups revealed that all control corneas were revascularized to the center with granuloma and retained a conjunctival phenotype. In contrast, in the experimental groups, 5 corneas became clear with either minimal or no vascularization; the rest had either mild peripheral (5) or total (3) vascularization and more cloudy stroma. Using monoclonal antibodies for epithelial markers and matrix components, we concluded that the success correlated with the return of a cornea-like epithelial phenotype and the preservation of the amniotic membrane, whereas the failure maintained a conjunctival epithelial phenotype and the amniotic membrane was either partially degraded or covered by host fibrovascular stroma. Measures taken to facilitate the former might prove this procedure clinically useful for ocular surface reconstruction.     

Differential effects of low and high concentrations of 4-aminopyridine on axonal conduction in normal and injured spinal cord

Laminins, the main components of basement membranes, are heterotrimers consisting of alpha, beta, and gamma polypeptide chains linked together by disulfide bonds. Laminins-1 and -2 are both composed of beta1 and gamma1 chains and differ from each other on their alpha chain, which is alpha1 and alpha2 for laminin-1 and -2, respectively. The present study shows that whereas laminins-1 and -2 are synthesized in the mouse developing lung and in epithelial-mesenchymal cocultures derived from it, epithelial and mesenchymal monocultures lose their ability to synthesize the laminin alpha1 chain. Synthesis of laminin alpha1 chain however returns upon re-establishment of epithelial-mesenchymal contact. Cell-cell contact is critical, since laminin alpha1 chain is not detected in monocultures exposed to coculture-conditioned medium or in epithelial-mesenchymal cocultures in which heterotypic cell-cell contact is prevented by an interposing filter. Immunohistochemical studies on cocultures treated with brefeldin A, an inhibitor of protein secretion, indicated both epithelial and mesenchymal cells synthesize laminin alpha1 chain upon heterotypic cell- cell contact. In a set of functional studies, embryonic lung explants were cultured in the presence of monoclonal antibodies to laminin alpha1, alpha2, and beta/gamma chains. Lung explants exposed to monoclonal antibodies to laminin alpha1 chain exhibited alterations in peribronchial cell shape and decreased smooth muscle development, as indicated by low levels of smooth muscle alpha actin and desmin. Taken together, our studies suggest that laminin alpha1 chain synthesis is regulated by epithelial-mesenchymal interaction and may play a role in airway smooth muscle development.     

Cell binding sequences in mouse laminin alpha1 chain

Laminin-1, a multifunctional glycoprotein of the basement membrane, consists of three different subunits, alpha1, beta1, and gamma1 chains. Previously, we used synthetic peptides to screen for biologically active sequences in the laminin alpha1 chain C-terminal globular domain (G domain) and identified several cell binding sequences (Nomizu, M., Kim, W. H., Yamamura, K., Utani, A., Song, S. Y., Otaka, A., Roller, P. P., Kleinman, H. K., and Yamada, Y. (1995) J. Biol. Chem. 270, 20583-20590). Here, we identify new cell binding sequences on the remainder of the laminin alpha1 chain by systematic peptide screening, using 208 overlapping synthetic peptides encompassing the central and N-terminal portions of the alpha1 chain. HT-1080 cell attachment activity to the peptides was evaluated using peptide-coated plastic substrates and peptide-conjugated Sepharose beads. Twenty five peptides showed cell attachment activities on either the peptide-coated plastic substrates and/or the peptide-conjugated Sepharose beads. A-13 (RQVFQVAYIIIKA) showed strongest cell attachment activity in both the assays. Cell attachment to 14 of the peptides was inhibited by heparin. EDTA and integrin antibodies inhibited cell adhesion to two of the peptides, A-13 and A-25, suggesting that these sites likely bind to integrins. These peptides inhibited cell attachment to laminin-1 but not to collagen I, suggesting these active sites are available on the intact molecule. Most of active sequences were localized on globular domains suggesting that these structures play a critical role in binding to cell-surface receptors.