Biomedical subjects

E Ruoslahti

Publications and source records attributed to E Ruoslahti.

At least 199 records · Page 11Linked to original sources

PubMed1985

Complete amino acid sequence of human vitronectin deduced from cDNA. Similarity of cell attachment sites in vitronectin and fibronectin.

cDNA clones for vitronectin, a cell adhesion-promoting plasma and tissue protein, were isolated from a lambda gt11 library containing cDNA inserts made from human liver mRNA. The library was screened with anti-vitronectin antibodies and the positive clones were further identified with synthetic oligonucleotide probes deduced from the partial amino acid sequence of vitronectin. Nucleotide sequence analysis showed that the largest insert was 1545 bp long and contained the whole sequence corresponding to plasma vitronectin. It showed that vitronectin contains the entire 44-amino acid somatomedin B peptide at its NH2 terminus and, near its COOH terminus, a 34-amino acid glycosaminoglycan binding site in which half of the amino acids are basic residues. Three potential carbohydrate attachment sites are present in the sequence. An Arg-Gly-Asp sequence, which has previously been shown to be the cell attachment site in fibronectin, was found in vitronectin immediately after the NH2-terminal somatomedin B sequence. No other homologies with fibronectin were found. The Arg-Gly-Asp sequence appears to constitute the cell attachment site of vitronectin, since it is in the region where we have previously localized the cell attachment site, its presence correlate with cell attachment activity among the insert-coded polypeptides, and because previous results have shown that synthetic peptides containing the Arg-Gly-Asp sequence inhibit the cell attachment function of vitronectin. The discovery of an Arg-Gly-Asp cell attachment site in a protein with a known cell attachment function emphasizes the general importance of this sequence in cell recognition.

Amino Acid Sequence↗

PubMed1985

The cell attachment determinant in fibronectin.

Fibronectin possesses a domain that interacts with cell surfaces. The ability of fibronectin to promote cell attachment can be duplicated with a short amino acid sequence, glycyl-L-arginyl-glycyl-L-aspartyl-L-serine, taken from that domain. The tripeptide Arg-Gly-Asp appears to be irreplaceable for maintenance of the activity of this peptide, whereas the serine residue can be replaced with some, but apparently not all, possible residues. This recognition sequence, or a closely related sequence, is present in a number of proteins other than fibronectin that interact with cells. These proteins include collagens, fibrinogen, thrombin, a bacterial surface protein, and two viral proteins, as well as discoidin-I, a protein implicated in the aggregation of Dictyostelium discoideum. A similar sequence is also repeated in some, but not all, fibronectin molecules, making it possible that some fibronectin molecules have more than a single cell attachment site. Synthetic peptides constructed from sequences taken from several of these other proteins have also been shown to promote cell attachment. The tripeptide sequence may, therefore, constitute an ancient cellular recognition mechanism common to many proteins.

Amino Acid Sequence↗

PubMed1985

Vitronectin--a major cell attachment-promoting protein in fetal bovine serum.

Bovine serum is a constituent of most media used for the culture of animal cells. The adhesion-promoting properties of serum are generally attributed to fibronectin, yet there have been frequent reports of other adhesion-promoting molecules in bovine serum. Using a technique in which adhesive proteins are visualized after separation by SDS-PAGE, we graphically confirm the presence of a second cell attachment protein in bovine serum and present the evidence that this molecule is the bovine equivalent of vitronectin. The molecular size of this protein is in the same range as the size of the adhesive human plasma protein, vitronectin. The bovine protein also shared with human vitronectin an affinity for glass, and it could be purified by a combination of glass bead and ion exchange chromatography. The isolated bovine protein had varying proportions of an 80 and a 65 kD polypeptide. It showed immunological cross-reactivity with anti-human vitronectin and with anti-human somatomedin B. Somatomedin B is a serum peptide which has a NH2-terminal sequence identical to that of human vitronectin. The identity of the bovine protein as vitronectin was established by showing that its NH2-terminal amino acid sequence is strongly homologous with those of human vitronectin and somatomedin B. Quantitation of the adhesive activities of fibronectin and vitronectin in bovine plasma and fresh serum showed that more activity is associated with vitronectin than with fibronectin. The preponderance of vitronectin was particularly clear in fetal bovine serum intended for cell culture. In various batches, cell attachment activity attributable to vitronectin was 8-16-fold greater than that of fibronectin, making vitronectin the main adhesive protein in routine cell culture media.

Amino Acid Sequence↗

PubMed1985

Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor.

Affinity chromatography was used to identify a putative cell surface receptor for fibronectin. A large cell-attachment-promoting fibronectin fragment was used as the affinity matrix, and specific elution was effected by using synthetic peptides containing the sequence Arg-Gly-Asp, which is derived from the cell recognition sequence in the fibronectin cell attachment site. A 140 kd protein was bound by the affinity matrix from octylglucoside extracts of MG-63 human osteosarcoma cells and specifically eluted with the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro. The 140 kd protein was labeled by cell surface specific radioiodination and became incorporated into liposomes at a high efficiency. Liposomes containing this protein showed specific affinity toward fibronectin-coated surfaces, and this binding could be selectively inhibited by the synthetic cell-attachment peptide but not by inactive peptides. Affinity chromatography on wheat germ agglutinin-Sepharose showed that the 140 kd protein is a glycoprotein and, in combination with the fibronectin fragment chromatography, gave highly enriched preparations of the 140 kd protein. These properties suggest that the 140 kd glycoprotein is a membrane-embedded cell surface protein directly involved in the initial step of cell adhesion to fibronectin substrates.

Autoradiography↗

PubMed1985

A 125/115-kDa cell surface receptor specific for vitronectin interacts with the arginine-glycine-aspartic acid adhesion sequence derived from fibronectin.

Affinity chromatography was used to identify a cell surface receptor for the adhesive protein vitronectin. Detergent extracts of human osteosarcoma (MG-63) cells were chromatographed on either vitronectin-Sepharose or Sepharose linked to the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro, which includes the fibronectin cell attachment sequence Arg-Gly-Asp. Two cell surface proteins with apparent molecular mass of 125 and 115 kDa bound to both columns and were specifically eluted with a solution containing the Gly-Arg-Gly-Asp-Ser-Pro peptide. These proteins could be incorporated into phosphatidylcholine liposomes and mediated the specific binding of these liposomes to vitronectin but not to fibronectin. In contrast, liposomes containing a previously identified 140-kDa fibronectin receptor, which interacts with the Arg-Gly-Asp sequence in fibronectin, did not bind to vitronectin. Thus, the fibronectin and vitronectin receptors each recognize the Gly-Arg-Gly-Asp-Ser-Pro peptide but exhibit mutually exclusive reactivities toward fibronectin and vitronectin. These receptors appear to belong to a family of proteins that mediate cell substratum adhesion via related but subtly different specificities.

Amino Acid Sequence↗

PubMed1985

The effect of Arg-Gly-Asp-containing peptides on fibrinogen and von Willebrand factor binding to platelets.

The Arg-Gly-Asp sequence resides in the cell attachment region of fibronectin. Arg-Gly-Asp-containing peptides support fibroblast attachment, inhibit fibroblast adhesion to fibronectin, and inhibit fibronectin binding to thrombin-stimulated platelets. In view of the similarities between the binding of fibronectin, fibrinogen, and von Willebrand factor to stimulated platelets, we have examined the effects of Arg-Gly-Asp-containing peptides on the interaction of these latter two adhesive proteins with platelets. Gly-Arg-Gly-Asp-Ser-Pro was used as a prototype peptide, and this hexapeptide inhibited fibrinogen binding to ADP and thrombin-stimulated platelets in the 10-200 microM range. The inhibition exceeded 90% at high concentrations of peptide and was observed in the presence of either calcium or magnesium. Platelet aggregation was also inhibited by the peptide in this dose range. The hexapeptide inhibited fibrinogen binding to platelets with receptors fixed in an exposed state, indicating direct interference with the ligand-platelet interaction. The peptide was 1/2 to 1/3rd as potent in inhibiting fibrinogen as fibronectin binding to platelets, but fibrinogen and von Willebrand factor binding were inhibited to an identical extent. Conservative amino acid substitutions for the arginine, glycine, or aspartic acid markedly reduced inhibitory activity and the Asp-Gly-Arg sequence was inactive. These results indicate that Arg-Gly-Asp-containing peptides can inhibit the binding of the three adhesive proteins to stimulated platelets, establishing a basic common feature between the interaction of these molecules with platelets.

Amino Acid Sequence↗

PubMed1985

Molecular cloning and sequence analysis of a chondroitin sulfate proteoglycan cDNA.

We report the identification and DNA sequence of a chondroitin sulfate proteoglycan core protein cDNA. A cDNA clone, pPG1, was selected from a rat yolk sac tumor poly(A)+RNA-derived cDNA library by using synthetic oligonucleotides predicted from the NH2-terminal peptide sequence of the mature chondroitin sulfate proteoglycan. The resulting sequence analysis demonstrated that the 874-base-pair pPG1 clone contained the complete coding region of the mature proteoglycan core protein as well as 5' and 3' flanking sequences. The 104 amino acid proteoglycan core protein sequence reveals that the core protein is composed of three regions, the most striking of which is the central 49 amino acid region composed of alternating serine and glycine residues. This region clearly functions as the acceptor site for the attachment of chondroitin sulfate side chains. The serine-glycine repeat region is flanked by a 14 amino acid NH2-terminal region identical to the NH2-terminal sequence of the proteoglycan obtained by amino acid sequencing and a 41 amino acid COOH-terminal region. RNA transfer blot hybridizations of poly(A)+ mRNA from rat yolk sac tumor cells with nick-translated pPG1 reveal a single mRNA of approximately equal to 1300 nucleotides. The possibility of detecting mRNAs and genomic sequences for other proteoglycans with a serine-glycine repeat by using this cDNA clone is discussed.

Amino Acid Sequence↗

PubMed1985

Detachment of cells from culture substrate by soluble fibronectin peptides.

The synthetic cell attachment-promoting peptides from fibronectin (Pierschbacher, M. D., and E. Ruoslahti, 1984, Nature (Lond.)., 309:30-33) were found to detach cultured cells from the substratum when added to the culture in a soluble form. Peptides ranging in length from tetrapeptide to heptapeptide and containing the active L-arginyl-glycyl-L-aspartic acid (Arg-Gly-Asp) sequence had the detaching activity, whereas a series of different peptides with chemically similar structures had no detectable effect on any of the test cells. The Arg-Gly-Asp-containing peptides caused detachment of various cell lines of different species and histogenetic origin. Studies with defined substrates showed that the active peptides could inhibit the attachment of cells to vitronectin in addition to fibronectin, indicating that vitronectin is recognized by cells through a similar mechanism as fibronectin. The peptides did not inhibit the attachment of cells to collagen. However, cells cultured on collagen-coated plastic for 24-36 h, as well as cells with demonstrable type I or type VI collagen in their matrix, were susceptible to the detaching effect of the peptides. These results indicate that the recognition mechanism(s) by which cells bind to fibronectinand vitronectin plays a major role in the substratum attachment of cells and that collagens may not be directly involved in cell-substratum adhesion. Since vitronectin is abundant in serum, it is probably an important component in mediating the attachment of cultured cells. The independence of the effects of the peptide on the presence of serum and the susceptibility of many different cell types to detachment by the peptide show that the peptides perturb an attachment mechanism that is intrinsic to the cells and fundamentally significant to their adhesion.

Amino Acid Sequence↗

PubMed1985

Effects of ricin A chain conjugates of monoclonal antibodies to human alpha-fetoprotein and placental alkaline phosphatase on antigen-producing tumor cells in culture.

Two tumor-associated proteins, alpha-fetoprotein (AFP) and placental alkaline phosphatase (PLAP), were investigated as target proteins for antibody:cytotoxin conjugates. An AFP-producing hepatoma cell line (HepG2) and a PLAP-producing cervical carcinoma cell line (SKGIIIa) were used as target cells. Both cell lines were equally susceptible to the toxic effects of intact ricin. Immunofluorescent studies showed that AFP could be detected at the surface of the HepG2 cells in a speckled distribution, while PLAP was uniformly distributed over the surface of the SKGIIIa cells. The anti-AFP ricin A chain conjugate was not cytotoxic to either cell line at low concentrations and killed both types of cells at high concentrations. The anti-PLAP conjugate at low concentrations was 100-fold more toxic than the anti-AFP conjugate to the PLAP-producing SKGIIIa cells. At high concentrations, it also killed both types of cells. The enhanced toxicity of the anti-PLAP conjugate to the SKGIIIa cells was inhibited by an excess of unconjugated anti-PLAP but not anti-AFP, indicating that the uptake of the conjugate depends on specific cell surface binding to the antigen. The indiscriminate toxicity observed at high concentrations of either conjugate was not inhibited by unconjugated antibody, suggesting that this effect depends on conjugate uptake independent of the identity of the antigen. These results emphasize the importance of the properties of the target antigen to the cytotoxic effects of antibody conjugates as well as the need for caution in experiments using high concentrations of conjugates. They suggest that PLAP may be a suitable target for immunotoxin therapy of human cancer.

Alkaline Phosphatase↗

PubMed1984

Domain structure of vitronectin. Alignment of active sites.

The structure of vitronectin, an adhesive protein isolated from human plasma, was studied by chemical fragmentation. Partial cleavage of vitronectin with cyanogen bromide in 70% formic acid generated four main fragments with masses of 53,000, 43,000, 35,000, and 12,000 daltons arising from both the 75- and 65-kDa vitronectin polypeptides and a 10-kDa fragment arising only from the 75-kDa polypeptide. By varying the reaction conditions, four BrCN cleavage sites and one acid cleavage site could be identified. The latter site gave rise to 40-, 32-, and 26-kDa fragments. The order of these fragments within the vitronectin polypeptides was determined by comparison of the NH2-terminal sequences of the polypeptides and their fragments, by further cleavage of the largest fragments with BrCN or 70% formic acid, and by assaying for heparin-binding and cell-attachment activities. The NH2-terminal sequences of the intact vitronectin polypeptides are the same and identical to a 44-amino acid serum peptide called somatomedin B, indicating that vitronectin may be the precursor of somatomedin B. The cell-attachment site appears to be located within approximately 5 kDa of the NH2 terminus, but it is distinct from the somatomedin B domain. The heparin-binding site is contained in the 12-kDa fragment near the COOH terminus. This fragment was shown to bind to a chondroitin sulfate proteoglycan in addition to heparin. The NH2-terminal amino acid sequence of this glycosaminoglycan-binding fragment is remarkably rich in basic amino acids. The NH2-terminal sequences of this and the other vitronectin fragments showed no homology with the amino acid sequence of the heparin-binding domain of fibronectin or other known sequences from fibronectin. These results show that the biological activities of vitronectin are located in distinct parts of both of the vitronectin polypeptides, which appear to be identical except for the presence of an additional 10-kDa fragment near or at the COOH terminus of the 75-kDa polypeptide.

Amino Acid Sequence↗

PubMed1984

Chondroitin/dermatan sulfate proteoglycan in human fetal membranes. Demonstration of an antigenically similar proteoglycan in fibroblasts.

A proteoglycan was isolated from fetal membranes which had been separated from human postpartum placenta. The glycosaminoglycan side chains (Mr = 55,000) were found to be composed of 75% chondroitin sulfate and 23% dermatan sulfate as determined by chondroitinase ABC or AC II digestion. NH2-terminal microsequencing of the intact proteoglycan revealed a single amino acid sequence of (sequence; see text) A rabbit antiserum raised against the intact proteoglycan reacted in sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting with Mr = 45,000 and 43,000 core polypeptides from chondroitinase-treated proteoglycan. Affinity-purified antibodies from this antiserum precipitated from human embryonic fibroblast culture fluid a proteoglycan which has an approximate Mr = 120,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This proteoglycan has on the average two polysaccharide side chains. As defined by chondroitinase digestion, these chains consist of 66% dermatan sulfate and 20% chondroitin sulfate. Digestion of the glycosaminoglycan with chondroitinase ABC converted the proteoglycan to a Mr = 45,000 major and a Mr = 43,000 minor core polypeptide. Tissue immunofluorescence localized the proteoglycan to interstitial matrices, suggesting that it is a product of mesenchymal cells. The methods we have devised for the purification of the fetal membrane proteoglycan in chemical amounts and the antibodies we have prepared against it will allow studies on the structural and functional properties of the proteoglycan and on the expression of immunologically cross-reactive proteoglycans by various cells and tissues.

Amino Acid Sequence↗

PubMed1984

Neurite-promoting factors and extracellular matrix components accumulating in vivo within nerve regeneration chambers.

The outgrowth of neurites from cultured neurons can be induced by the extracellular matrix glycoproteins, fibronectin and laminin, and by polyornithine-binding neurite-promoting factors (NPFs) derived from culture media conditioned by Schwann, or other cultured cells. We have examined the occurrence of fibronectin, laminin and NPFs during peripheral nerve regeneration in vivo. A previously established model of peripheral nerve regeneration was used in which a transected rat sciatic nerve regenerates through a silicone chamber bridging a 10 mm interstump gap. The distribution of fibronectin and laminin during regeneration was assessed by indirect immunofluorescence. Seven days after nerve transection the regenerating structure within the chamber consisted primarily of a fibrous matrix which stained with anti-fibronectin but not anti-laminin. At 14 days, cellular outgrowths from the proximal and distal stumps (along which neurites grow) had entered the fibronectin-containing matrix, consistent with a role of fibronectin in promoting cell migration. Within these outgrowths non-vascular as well as vascular cells stained with anti-fibronectin and anti-laminin. Within the degenerated distal nerve segment, cell characteristic of Bungner bands (rows of Schwann cells along which regenerating neurites extend) stained with anti-fibronectin and laminin. The fluid surrounding the regenerating nerve was found to contain NPF activity for cultured ciliary ganglia neurons which markedly increased during the period of neurite growth into the chamber. In previous studies using this particular neurite-promoting assay, laminin but to a much lesser extent fibronectin also promoted neurite outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1984

Fibronectin in cell adhesion and invasion.

Fibronectin plays a major role in the adhesion of many cell types. The extent of cell adhesion in vitro is related not only to the ability of the cells to interact with matrix-bound fibronectin, when it is present, but also to the synthesis or lack of synthesis of fibronectin by the cells, and to the lack of deposition of synthesized fibronectin into an insoluble matrix surrounding the cells. Many malignant cells, regardless of whether they synthesize subnormal or normal amounts of fibronectin, fail to deposit that fibronectin into a surrounding insoluble matrix. The lack of fibronectin around such cells appears to reflect a general absence of extracellular matrix since other matrix components, such as collagen, laminin, and heparan sulfate proteoglycan, are concomitantly missing. Cells that lack their own cell surface fibronectin due either to lack of deposition or to lack of synthesis can nevertheless adhere to insoluble fibronectin matrices elaborated by other cells. These cellular characteristics appear to be associated with cell migration in vivo during embryogenesis, and the same characteristics may enhance the invasive potential of malignant cells. The remarkable effects that fibronectin has on cellular adhesion and the association of lack of extracellular matrix components with poorly differentiated and highly metastatic tumors in vivo mandates that more be learned about the molecular and cellular details of the interactions of cells with their surrounding matrix. Important information concerning tumor invasion will parallel such an understanding and may eventually become the basis for therapeutic approaches.

Animals↗

PubMed1984

Clonal tumorigenic endodermal cell lines producing basement membrane components.

Tumors were developed from the mouse teratocarcinoma-derived endodermal cell line PF HR-9 by subcutaneous injections in syngeneic mice of large numbers of cells previously cultured for several years at high cell density. Cell cultures were established from the tumors and the cells were cloned. The cloned sublines were highly malignant in vivo and tumor metastases were occasionally observed. The tumors contained abundant extracellular material, which was distinctly laminated and contained type IV collagen, laminin, and heparan sulfate proteoglycan. The tumorigenic sublines were also shown to have retained markers of the original cells, such as the cytoskeletal proteins Endo A and B. These cell lines should be useful for biosynthetic studies on basement membrane and cytoskeletal components and the tumors for isolation of these macromolecules and their mRNAs.

Animals↗

PubMed1984

Variants of the cell recognition site of fibronectin that retain attachment-promoting activity.

A tetrapeptide sequence, Arg-Gly-Asp-Ser, is the minimal structure recognized by cells in the large, adhesive glycoprotein fibronectin. We now have defined the structural requirements for this cell recognition site by testing several synthetic variants of the active tetrapeptide sequence. The conservative substitutions of lysine for arginine, alanine for glycine, or glutamic acid for aspartic acid each resulted in abrogation of the cell attachment-promoting activity characteristic of the natural sequence. However, in the position of the serine residue, some alterations were compatible with activity. Assay of peptides containing the structure Arg-Gly-Asp-X (where X = another amino acid residue) showed that an Arg-Gly-Asp-Val sequence predicted to be present in some, but not all, fibronectin molecules as a result of alternative RNA splicings could potentially create a second cell attachment site in those fibronectin polypeptide chains carrying that sequence. Other proteins with potentially active Arg-Gly-Asp-X sequences include several proteins that are known to interact with the cell surface. Among these are various types of collagens, thrombin, and discoidin, a slime-mold protein that may be involved in cell aggregation. The result presented here show that the arginine, glycine, and aspartic acid residues are absolutely required for the cell recognition, and that the surrounding amino acids may play a role in the expression of cell attachment activity in fibronectin and other proteins having this sequence. We suggest, based on these data, that this recognition mechanism may be common to a number of biological systems.

Amino Acid Sequence↗

PubMed1984

Adhesion of platelets to laminin in the absence of activation.

The binding of platelets to components in the subendothelial matrix is an initial event in hemostasis and thrombosis. The glycoprotein components of the matrix are considered important in this interaction. Of these, collagen binds and activates platelets and induces their aggregation. In this study we demonstrate that substrate-bound laminin causes time- and concentration-dependent adherence of human platelets to the substrate. The binding of platelets to laminin was found to be similar in some respects, but different in others, to their binding to surfaces coated with fibronectin or collagen. The binding of platelets to laminin or fibronectin was not associated with their activation under conditions in which type I collagen activates the platelets as measured by [14C]serotonin secretion. Platelets bound to laminin and fibronectin differed in their appearance; they remained rounded on laminin whereas they flattened completely on fibronectin. Binding of platelets to fibronectin, but not laminin, is inhibited by a recently described peptide (Pierschbacher, M., and E. Ruoslahti, 1984, Nature (Lond.), 309:30-33) containing the cell-attachment tetrapeptide sequence of fibronectin, which suggests that separate receptors exist for laminin and fibronectin. These studies establish laminin as a platelet-binding protein and suggest that laminin can contribute to the adhesiveness of exposed tissue matrices to platelets. Since laminin and fibronectin do not activate platelets, whereas collagen does, and laminin differs from fibronectin in that it does not induce spreading of the attached platelets, all three proteins appear to confer different signals to the platelets. Some of these may be related to platelet functions other than those necessary for the formation of a hemostatic plug.

Blood Platelets↗

PubMed1983

gamma-Glutamyl transpeptidase in human amniotic fluid and in fetal tissues.

The activity of gamma-glutamyl transpeptidase (GGT) was measured in 360 amniotic fluid samples from second and third trimester pregnancies with fetal and maternal disorders. The enzyme level was within the normal range in all but one of the 18 second trimester pregnancies with congenital malformations. On cellulose acetate electrophoresis, GGT fractionated as an alpha 1-band in all mid-trimester samples tested. Highly elevated GGT values were found in five samples from the third trimester gestations with fetal gastroschisis, hydrops fetalis and Meckel's syndrome. The enzyme in these samples showed alpha 1-mobility while most of the third trimester amniotic fluids contained GGT with beta-mobility. Considerable GGT activity was observed in several fetal tissues that are in contact with the amniotic fluid. Highest levels were found in meconium and bile from a second trimester fetus. The total activity and electrophoretic mobility of GGT in late amniotic fluid and in urine from newborns was similar.

Amniotic Fluid↗

PubMed1983

Human laminin isolated in a nearly intact, biologically active form from placenta by limited proteolysis.

A protein with properties of laminin has been isolated from human placental extracts by using monoclonal antibodies. Placental tissue was extracted with 0.5 M NaCl and high molecular weight proteins were isolated from the extract by salt precipitation and gel filtration on Sepharose 6B. The resulting protein fraction which contained material cross-reactive with anti-sera to rat laminin was used as immunogen to prepare hybridomas. Thirteen hybrids produced antibodies which reacted with basement membrane-associated antigens in indirect immunofluorescence of tissues. One of these, 4E10, was characterized in detail. This monoclonal antibody reacted with human laminin as shown by several lines of evidence. Immunoprecipitation from metabolically labeled culture media of a human amniotic epithelial cell line with the 4E10 antibody followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed polypeptides with Mr similar to those of rat laminin. Immunochromatography of placental extracts obtained by limited pepsin digestion yielded material with main polypeptides at 160 and 130 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction. These peptic fragments cross-reacted with rat laminin in immunodiffusion and enzyme immunoassay, and a polyclonal antiserum against the fragments reacted with basement membranes in tissues in a manner identical with the 4E10 antibody. Electron microscopic images of the human peptic fragments showed structures similar to the cross-shaped images of murine laminins, although the short arms were truncated to various degrees or even absent. The isolated peptic fragments also displayed biological activity similar to that of murine laminins in that the outgrowth of neurites by neuronal cells was promoted on plates coated with the fragments.

Antibodies, Monoclonal↗