OBJECTIVE

Patients with schizophrenia frequently present with negative symptoms and cognitive impairments for which no effective treatments are known. Agents that act at the glycine site of the N-methyl-D-aspartic acid (NMDA) glutamatergic receptor have been suggested as promising treatments for moderate to severe negative symptoms and cognitive impairments.

METHODS

The Cognitive and Negative Symptoms in Schizophrenia Trial (CONSIST) was a 16-week double-blind, double-dummy, parallel group, randomized clinical trial of adjunctive glycine, D-cycloserine, or placebo conducted at four sites in the United States and one site in Israel. The participants were 157 inpatients and outpatients who met DSM-IV criteria for schizophrenia or schizoaffective disorder and retrospective and prospective criteria for moderate to severe negative symptoms without marked positive, depressive, or extrapyramidal symptoms. The primary outcome measures were the average "rate of change" of Scale for the Assessment of Negative Symptoms (SANS) total scores and change in the average cognitive domain z scores.

RESULTS

There were no significant differences in change in the SANS total score between glycine and placebo subjects or D-cycloserine and placebo subjects. A prespecified test for the site-by-treatment-by-time interaction was significant in post hoc tests. One site had greater reduction in the SANS total score for patients receiving D-cycloserine relative to patients receiving placebo. A second site had greater reduction in the SANS total score for placebo patients compared with glycine patients. There were no significant differences between glycine and placebo or D-cycloserine and placebo subjects on the average cognition z score.

CONCLUSIONS

The study results suggest that neither glycine nor D-cycloserine is a generally effective therapeutic option for treating negative symptoms or cognitive impairments.

The effect of C-terminal tyrosine phosphorylation on molecular motions in the Src kinases Hck and c-Src is investigated by molecular dynamics simulations. The SH2 and SH3 domains of the inactive kinases are seen to be tightly coupled by the connector between them, impeding activation. Dephosphorylation of the tail reduces the coupling between the SH2 and SH3 domains in the simulations, as does replacement of connector residues with glycine. A mutational analysis of c-Src expressed in Schizosaccharomyces pombe demonstrates that replacement of residues in the SH2-SH3 connector with glycine activates c-Src. The SH2-SH3 connector appears to be an inducible "snap lock" that clamps the SH2 and SH3 domains upon tail phosphorylation, but which allows flexibility when the tail is released.

We have previously established a minimalist approach to antibody engineering by using a phage-displayed framework to support complementarity determining region (CDR) diversity restricted to a binary code of tyrosine and serine. Here, we systematically augmented the original binary library with additional levels of diversity and examined the effects. The diversity of the simplest library, in which only heavy chain CDR positions were randomized by the binary code, was expanded in a stepwise manner by adding diversity to the light chain, by diversifying non-paratope residues that may influence CDR conformations, and by adding additional chemical diversity to CDR-H3. The additional diversity incrementally improved the affinities of antibodies raised against human vascular endoethelial growth factor and the structure of an antibody-antigen complex showed that tyrosine side-chains are sufficient to mediate most of the interactions with antigen, but a glycine residue in CDR-H3 was critical for providing a conformation suitable for high-affinity binding. Using new high-throughput procedures and the most complex library, we produced multiple high-affinity antibodies with dissociation constants in the single-digit nanomolar range against a wide variety of protein antigens. Thus, this fully synthetic, minimalist library has essentially recapitulated the capacity of the natural immune system to generate high-affinity antibodies. Libraries of this type should be highly useful for proteomic applications, as they minimize inherent complexities of natural antibodies that have hindered the establishment of high-throughput procedures. Furthermore, analysis of a large number of antibodies derived from these well-defined and simplistic libraries allowed us to uncover statistically significant trends in CDR sequences, which provide valuable insights into antibody library design and into factors governing protein-protein interactions.

Mass spectrometry investigations of partially purified Campylobacter jejuni protein PEB3 showed it to be partially modified with an Asn-linked glycan with a mass of 1406 Da and composed of one hexose, five N-acetylhexosamines and a species of mass 228 Da, consistent with a trideoxydiacetamidohexose. By means of soybean lectin affinity chromatography, a mixture of glycoproteins was obtained from a glycine extract, and two-dimensional gel proteomics analysis led to the identification of at least 22 glycoproteins, predominantly annotated as periplasmic proteins. Glycopeptides were prepared from the glycoprotein mixture by Pronase digestion and gel filtration. The structure of the glycan was determined by using nano-NMR techniques to be GalNAc-alpha1,4-GalNAc-alpha1,4-[Glcbeta1,3-]GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac-beta1,N-Asn-Xaa, where Bac is bacillosamine, 2,4-diacetamido-2,4,6-trideoxyglucopyranose. Protein glycosylation was abolished when the pglB gene was mutated, providing further evidence that the enzyme encoded by this gene is responsible for formation of the glycopeptide N-linkage. Comparison of the pgl locus with that of Neisseria meningitidis suggested that most of the homologous genes are probably involved in the biosynthesis of bacillosamine.

Little is known about the function of flavivirus nonstructural protein NS2A. Two forms of NS2A are found in yellow fever virus-infected cells. Full-length NS2A (224 amino acids) is the product of cleavage at the NS1/2A and NS2A/2B sites. NS2Aalpha, a C-terminally truncated form of 190 amino acids, results from partial cleavage by the viral NS2B-3 serine protease at the sequence QK /T within NS2A. Exchange of serine for lysine at this site (QKT->>QST) blocks the production of both NS2Aalpha and infectious virus. The present study reveals that this defect is not at the level of RNA replication. Despite normal structural region processing, infectious particles containing genome RNA and capsid protein were not released from cells transfected with the mutant RNA. Nevertheless, production of subviral prM/M- and E-containing particles was unimpaired. The NS2A defect could be complemented in trans by providing NS1-2A or NS1-2Aalpha. However, trans complementation was not observed when the C-terminal lysine of NS1-2Aalpha was replaced with serine. In addition to true reversions, NS2Aalpha cleavage site mutations could be suppressed by two classes of second-site changes. The first class consisted of insertions at the NS2Aalpha cleavage site that restored its basic character and cleavability. A second class of suppressors occurred in the NS3 helicase domain, in which NS3 aspartate 343 was replaced with an uncharged residue (either valine, alanine, or glycine). These mutations in NS3 restored infectious-virus production in the absence of cleavage at the mutant NS2Aalpha site. Taken together, our results reveal an unexpected role for NS2A and NS3 in the assembly and/or release of infectious flavivirus particles.

Eukaryotic cells compartmentalize biochemical processes in different organelles, often relying on metabolic cycles to shuttle reducing equivalents across intracellular membranes. NADPH serves as the electron carrier for the maintenance of redox homeostasis and reductive biosynthesis, with separate cytosolic and mitochondrial pools providing reducing power in each respective location. This cellular organization is critical for numerous functions but complicates analysis of metabolic pathways using available methods. Here we develop an approach to resolve NADP(H)-dependent pathways present within both the cytosol and the mitochondria. By tracing hydrogen in compartmentalized reactions that use NADPH as a cofactor, including the production of 2-hydroxyglutarate by mutant isocitrate dehydrogenase enzymes, we can observe metabolic pathway activity in these distinct cellular compartments. Using this system we determine the direction of serine/glycine interconversion within the mitochondria and cytosol, highlighting the ability of this approach to resolve compartmentalized reactions in intact cells.

Heterodimeric integrin adhesion receptors regulate cell migration, survival and differentiation in metazoa by communicating signals bi-directionally across the plasma membrane. Protein engineering and mutagenesis studies have suggested that the dissociation of a complex formed by the single-pass transmembrane (TM) segments of the alpha and beta subunits is central to these signalling events. Here, we report the structure of the integrin alphaIIbbeta3 TM complex, structure-based site-directed mutagenesis and lipid embedding estimates to reveal the structural event that underlies the transition from associated to dissociated states, that is, TM signalling. The complex is stabilized by glycine-packing mediated TM helix crossing within the extracellular membrane leaflet, and by unique hydrophobic and electrostatic bridges in the intracellular leaflet that mediate an unusual, asymmetric association of the 24- and 29-residue alphaIIb and beta3 TM helices. The structurally unique, highly conserved integrin alphaIIbbeta3 TM complex rationalizes bi-directional signalling and represents the first structure of a heterodimeric TM receptor complex.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated epithelial Cl- channel that, when defective, causes cystic fibrosis. Screening of a collection of 100,000 diverse small molecules revealed four novel chemical classes of CFTR inhibitors with Ki < 10 microM, one of which (glycine hydrazides) had many active structural analogues. Analysis of a series of synthesized glycine hydrazide analogues revealed maximal inhibitory potency for N-(2-naphthalenyl) and 3,5-dibromo-2,4-dihydroxyphenyl substituents. The compound N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide (GlyH-101) reversibly inhibited CFTR Cl- conductance in <1 min. Whole-cell current measurements revealed voltage-dependent CFTR block by GlyH-101 with strong inward rectification, producing an increase in apparent inhibitory constant Ki from 1.4 microM at +60 mV to 5.6 microM at -60 mV. Apparent potency was reduced by lowering extracellular Cl- concentration. Patch-clamp experiments indicated fast channel closures within bursts of channel openings, reducing mean channel open time from 264 to 13 ms (-60 mV holding potential, 5 microM GlyH-101). GlyH-101 inhibitory potency was independent of pH from 6.5-8.0, where it exists predominantly as a monovalent anion with solubility approximately 1 mM in water. Topical GlyH-101 (10 microM) in mice rapidly and reversibly inhibited forskolin-induced hyperpolarization in nasal potential differences. In a closed-loop model of cholera, intraluminal GlyH-101 (2.5 microg) reduced by approximately 80% cholera toxin-induced intestinal fluid secretion. Compared with the thiazolidinone CFTR inhibitor CFTR(inh)-172, GlyH-101 has substantially greater water solubility and rapidity of action, and a novel inhibition mechanism involving occlusion near the external pore entrance. Glycine hydrazides may be useful as probes of CFTR pore structure, in creating animal models of CF, and as antidiarrheals in enterotoxic-mediated secretory diarrheas.

Several families with an early-onset form of familial Alzheimer's disease have been found to harbour mutations at a specific codon (717) of the gene for the beta-amyloid precursor protein (APP) on chromosome 21. We now report, a novel base mutation in the same exon of the APP gene which co-segregates in one family with presenile dementia and cerebral haemorrhage due to cerebral amyloid angiopathy. The mutation results in the substitution of alanine into glycine at codon 692. These results suggest that the clinically distinct entities, presenile dementia and cerebral amyloid angiopathy, can be caused by the same mutation in the APP gene.

The pituitary hormone thyrotropin stimulates the function, expression of differentiation and growth of thyrocytes by cyclic AMP-dependent mechanisms. Tissue hyperplasia and hyperthyroidism are therefore expected to result when activation of the adenylyl cyclase-cAMP cascade is unregulated. This is observed in several situations, including when somatic mutations impair the GTPase activity of the G protein Gsa (ref 6, 7). Such a mechanism is probably responsible for the development of a minority of monoclonal hyperfunctioning thyroid adenomas. Here we identify somatic mutations in the carboxy-terminal portion of the third cytoplasmic loop of the thyrotropin receptor in three out of eleven hyperfunctioning thyroid adenomas. These mutations are restricted to tumour tissue and involve two different residues (aspartic acid at position 619 to glycine in two cases, and alanine at position 623 to isoleucine in one case). The mutant receptors confer constitutive activation of adenylyl cyclase when tested by transfection in COS cells. This shows that G-protein-coupled receptors are susceptible to constitutive activation by spontaneous somatic mutations and may thus behave as proto-oncogenes.

The role of myristylation, a fatty acid modification of nascent polypeptides, in the assembly and intracellular transport of D-type retroviral capsids was investigated through the use of oligonucleotide-directed mutagenesis. Myristic acid is normally esterified through an amide linkage to a glycine residue at the amino terminus of the Mason-Pfizer monkey virus gag gene products. Mutant pA-1, which has a codon for valine substituted for that of the normally myristylated glycine, is completely noninfectious. While the mutant gag polyprotein precursors are synthesized at normal levels, they are not myristylated and are not cleaved to the mature virion proteins. No extracellular virus particles are released from mutant pA-1-infected cells, but intracytoplasmic A-type particles (capsids) accumulate in the cytoplasm. Since none of the intracellular capsids can be found associated with the plasma membrane, these results strongly suggest that myristylation is a critical signal for intracytoplasmic transport of completed viral capsids to their normal site of budding and release.

Nuclear pore complexes reside in the nuclear envelope of eukaryotic cells and mediate the nucleocytoplasmic exchange of macromolecules. Traffic is regulated by mobile transport receptors that target their cargo to the central translocation channel, where phenylalanine-glycine-rich repeats serve as binding sites. The structural analysis of the nuclear pore is a formidable challenge given its size, its location in a membranous environment and its dynamic nature. Here we have used cryo-electron tomography to study the structure of nuclear pore complexes in their functional environment, that is, in intact nuclei of Dictyostelium discoideum. A new image-processing strategy compensating for deviations of the asymmetric units (protomers) from a perfect eight-fold symmetry enabled us to refine the structure and to identify new features. Furthermore, the superposition of a large number of tomograms taken in the presence of cargo, which was rendered visible by gold nanoparticles, has yielded a map outlining the trajectories of import cargo. Finally, we have performed single-molecule Monte Carlo simulations of nuclear import to interpret the experimentally observed cargo distribution in the light of existing models for nuclear import.

Shc proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to Ras. The p46shc and p52shc isoforms share a C-terminal SH2 domain, a proline- and glycine-rich region (collagen homologous region 1; CH1) and a N-terminal PTB domain. We have isolated cDNAs encoding for a third Shc isoform, p66shc. The predicted amino acid sequence of p66shc overlaps that of p52shc and contains a unique N-terminal region which is also rich in glycines and prolines (CH2). p52shc/p46shc is found in every cell type with invariant reciprocal relationship, whereas p66shc expression varies from cell type to cell type. p66shc differs from p52shc/p46shc in its inability to transform mouse fibroblasts in vitro. Like p52shc/p46shc, p66shc is tyrosine-phosphorylated upon epidermal growth factor (EGF) stimulation, binds to activated EGF receptors (EGFRs) and forms stable complexes with Grb2. However, unlike p52shc/p46shc it does not increase EGF activation of MAP kinases, but inhibits fos promoter activation. The isolated CH2 domain retains the inhibitory effect of p66shc on the fos promoter. p52shc/p46shc and p66shc, therefore, appear to exert different effects on the EGFR-MAP kinase and other signalling pathways that control fos promoter activity. Regulation of p66shc expression might, therefore, influence the cellular response to growth factors.

A better understanding of human hepatocellular carcinoma (HCC) pathogenesis at the molecular level will facilitate the discovery of tumor-initiating events. Transcriptome sequencing revealed that adenosine-to-inosine (A→I) RNA editing of AZIN1 (encoding antizyme inhibitor 1) is increased in HCC specimens. A→I editing of AZIN1 transcripts, specifically regulated by ADAR1 (encoding adenosine deaminase acting on RNA-1), results in a serine-to-glycine substitution at residue 367 of AZIN1, located in β-strand 15 (β15) and predicted to cause a conformational change, induced a cytoplasmic-to-nuclear translocation and conferred gain-of-function phenotypes that were manifested by augmented tumor-initiating potential and more aggressive behavior. Compared with wild-type AZIN1 protein, the edited form has a stronger affinity to antizyme, and the resultant higher AZIN1 protein stability promotes cell proliferation through the neutralization of antizyme-mediated degradation of ornithine decarboxylase (ODC) and cyclin D1 (CCND1). Collectively, A→I RNA editing of AZIN1 may be a potential driver in the pathogenesis of human cancers, particularly HCC.

The nature of cell wall proteins is as varied as the many functions of plant cell walls. With the exception of glycine-rich proteins, all are glycosylated and contain hydroxyproline (Hyp). Again excepting glycine-rich proteins, they also contain highly repetitive sequences that can be shared between them. The majority of cell wall proteins are cross-linked into the wall and probably have structural functions, although they may also participate in morphogenesis. On the other hand, arabinogalactan proteins are readily soluble and possibly play a major role in cell-cell interactions during development. The interactions of these proteins between themselves and with other wall components is still unknown, as is how wall components are assembled. The possible functions of cell wall proteins are suggested based on repetitive sequence, localization in the plant body, and the general morphogenetic pattern in plants.

Previous work has shown that some cancer cells are highly dependent on serine/glycine uptake for proliferation. Although serine and glycine can be interconverted and either might be used for nucleotide synthesis and one-carbon metabolism, we show that exogenous glycine cannot replace serine to support cancer cell proliferation. Cancer cells selectively consumed exogenous serine, which was converted to intracellular glycine and one-carbon units for building nucleotides. Restriction of exogenous glycine or depletion of the glycine cleavage system did not impede proliferation. In the absence of serine, uptake of exogenous glycine was unable to support nucleotide synthesis. Indeed, higher concentrations of glycine inhibited proliferation. Under these conditions, glycine was converted to serine, a reaction that would deplete the one-carbon pool. Providing one-carbon units by adding formate rescued nucleotide synthesis and growth of glycine-fed cells. We conclude that nucleotide synthesis and cancer cell proliferation are supported by serine--rather than glycine--consumption.

In mammalian tissues the C-terminal amide structure has been found to occur only in neuroactive or hormonally-active peptides. About half known neuropeptide and peptide hormones have this unique chemical feature. Using a chemical detection method, a search for previously unknown peptides that possess the C-terminal amide structure in extracts of brain and intestine was carried out and a number of novel neuropeptides and hormonal peptides, designated neuropeptide Y, PHI, peptide YY, galanin and neuropeptide K were isolated. We recently performed a similar search in porcine pancreas and found a high concentration of a peptide having a glycine amide at its C-terminus. Here we report the isolation, primary structure and biological activity of this novel peptide. The 49-residue peptide strongly inhibits glucose-induced insulin release from the isolated perfused pancreas and was therefore named pancreastatin. It may be important in the regulation of insulin secretion and in the pathogenesis and treatment of diabetes mellitus.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition in which motoneurons of the spinal cord and motor cortex die, resulting in progressive paralysis. This condition has no cure and results in eventual death, usually within 1-5 years of diagnosis. Although the specific etiology of ALS is unknown, 20% of familial cases of the disease carry mutations in the gene encoding Cu/Zn superoxide dismutase-1 (SOD1). Transgenic mice overexpressing human mutant SOD1 have a phenotype and pathology that are very similar to that seen in human ALS patients. Here we show that treatment with arimoclomol, a coinducer of heat shock proteins (HSPs), significantly delays disease progression in mice expressing a SOD1 mutant in which glycine is substituted with alanine at position 93 (SOD1(G93A)). Arimoclomol-treated SOD1(G93A) mice show marked improvement in hind limb muscle function and motoneuron survival in the later stages of the disease, resulting in a 22% increase in lifespan. Pharmacological activation of the heat shock response may therefore be a successful therapeutic approach to treating ALS, and possibly other neurodegenerative diseases.

Processing of the amyloid precursor protein (APP) by beta- and gamma-secretases leads to the generation of amyloid-beta (Abeta) peptides with varying lengths. Particularly Abeta42 contributes to cytotoxicity and amyloid accumulation in Alzheimer's disease (AD). However, the precise molecular mechanism of Abeta42 generation has remained unclear. Here, we show that an amino-acid motif GxxxG within the APP transmembrane sequence (TMS) has regulatory impact on the Abeta species produced. In a neuronal cell system, mutations of glycine residues G29 and G33 of the GxxxG motif gradually attenuate the TMS dimerization strength, specifically reduce the formation of Abeta42, leave the level of Abeta40 unaffected, but increase Abeta38 and shorter Abeta species. We show that glycine residues G29 and G33 are part of a dimerization site within the TMS, but do not impair oligomerization of the APP ectodomain. We conclude that gamma-secretase cleavages of APP are intimately linked to the dimerization strength of the substrate TMS. The results demonstrate that dimerization of APP TMS is a risk factor for AD due to facilitating Abeta42 production.

A cell motility-stimulating factor has been isolated, purified, and partially characterized from the serum-free conditioned medium of human A2058 melanoma cells. We term this activity "autocrine motility factor" (AMF). AMF has the properties of a protein with an estimated size of 55 kDa. At concentrations of 10 nM or less, AMF stimulated the random or directed motility of the producer cells. However, AMF is not an attractant for neutrophils. Amino acid analysis of the purified AMF protein revealed a high content of serine, glycine, glutamic acid, and aspartic acid residues. The activity of AMF was not replaced or blocked by known growth factors such as epidermal growth factor or type beta transforming growth factor. Mechanistic studies showed that AMF stimulated the incorporation of [3H]methyl into cell membrane phospholipids after incubation with [methyl-3H]methionine with a sustained increase in the methylation of phosphatidyldimethylethanolamine to phosphatidylcholine. In contrast, AMF did not affect the incorporation of [1,2-14C]choline into phosphatidylcholine. AMF was produced in large amounts by three different clones of ras oncogene-transfected metastatic NIH 3T3 cells but not by the nontransformed parental cells. AMF may play a major role in the local invasive behavior of tumor cells and may also facilitate the concerted invasion by groups of tumor cells.

It has been shown previously that Escherichia coli accumulates endogenously synthesized trehalose under osmotic stress. We report here that E. coli contained an osmotically regulated trehalose-phosphate synthase which utilized UDP-glucose and glucose 6-phosphate as substrates. In the wild type, the synthase was induced by growth in glucose-mineral medium of elevated osmotic strength and the synthase itself was strongly stimulated by K+ and other monovalent cations. A laboratory strain which expressed the synthase at a high constitutive level was found. GalU mutants, defective in synthesis of UDP-glucose, did not accumulate trehalose. Two genes governing the synthase were identified and named otsA and otsB (osmoregulatory trehalose synthesis). They mapped near 42 min in the flbB-uvrC region. Mutants with an otsA-lacZ or otsB-lacZ operon fusion displayed osmotically inducible beta-galactosidase activity; i.e., the activity was increased fivefold by growth in medium of elevated osmotic strength. Mutants unable to synthesize trehalose (galU, otsA, and otsB) were osmotically sensitive in glucose-mineral medium. But an osmotically tolerant phenotype was restored in the presence of glycine betaine, which also partially repressed the synthesis of synthase in the wild type and of beta-galactosidase in ots-lacZ fusion mutants.

The cellular receptor for human urokinase-type plasminogen activator (u-PAR) is shown by several independent criteria to be a true member of a family of integral membrane proteins, anchored to the plasma membrane exclusively by a COOH-terminal glycosyl-phosphatidylinositol moiety. 1) Amino acid analysis of u-PAR after micropurification by affinity chromatography and N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of 2-3 mol of ethanolamine/mol protein. 2) Membrane-bound u-PAR is efficiently released from the surface of human U937 cells by trace amounts of purified bacterial phosphatidylinositol-specific phospholipase C. This soluble form of u-PAR retains the binding specificity toward both u-PA and its amino-terminal fragment holding the receptor-binding domain. 3) Treatment of purified u-PAR with phosphatidylinositol-specific phospholipase C or mild alkali completely alters the hydrophobic properties of the receptor as judged by temperature-induced detergent-phase separation and charge-shift electrophoresis. 4) Biosynthetic labeling of u-PAR was obtained with [3H]ethanolamine and myo-[3H]inositol. 5) Finally, comparison of amino acid compositions derived from cDNA sequence and amino acid analysis shows that a polypeptide of medium hydrophobicity is excised from the COOH terminus of the nascent u-PAR. A similar proteolytic processing has been reported for other proteins that are linked to the plasma membrane by a glycosyl-phosphatidylinositol membrane anchor.

The amino acid sequence RGD (arginine-glycine-aspartic acid) is highly conserved in the VP1 protein of foot-and-mouth disease virus (FMDV), despite being situated in the immunodominant hypervariable region between amino acids 135 and 160. RGD-containing proteins are known to be important in promoting cell attachment in several different systems, and we report here that synthetic peptides containing this sequence are able to inhibit attachment of the virus to baby hamster kidney (BHK) cells. Inhibition was dose-dependent and could be reversed on removal of the peptide. A synthetic peptide corresponding to a portion of the same hypervariable region but not containing the RGD sequence did not inhibit virus attachment under the same conditions. Antibody against the RGD region of VP1 blocked attachment of the virus to BHK cells, and neutralizing monoclonal antibodies, which neutralize virus by preventing cell attachment, were blocked by RGD-containing peptides from binding virus in an ELISA test. Cleavage of the C-terminal region of virus VP1 in situ with proteolytic enzymes reduced cell attachment, and antiserum against a peptide corresponding to this region was also able to inhibit attachment of virus to BHK cells. These results indicate that the amino acid sequence RGD at positions 145 to 147 and amino acids from the C-terminal region of VP1 (positions 203 to 213) contribute to the cell attachment site on FMDV for BHK cells.