Site-specific Monoclonal Antibodies Research Articles

Monoclonal Antibodies as Probes to Study Ligand-Induced Conformations of Troponin Subunits.

Striated muscle contraction and relaxation is regulated by Ca2+ at the myofilament level via conformational modulations of the troponin complex. To understand the structure–function relationship of troponin in normal muscle and in myopathies, it is necessary to study the functional effects of troponin isoforms and mutations at the level of allosteric conformations of troponin subunits. Traditional methodologies assessing such conformational studies are laborious and require significant amounts of purified protein, while many current methodologies require non-physiological conditions or labeling of the protein, which may affect their physiological conformation and function. To address these issues, we developed a novel approach using site-specific monoclonal antibodies (mAb) as molecular probes to detect and monitor conformational changes of proteins. Here, we present examples for its application in studies of two subunits of troponin: the Ca2+-binding subunit, TnC, and the tropomyosin-binding/thin filament-anchoring subunit, TnT. Studies using a high-throughput microplate assay are compared with that using localized surface plasmon resonance (LSPR) to demonstrate the effectiveness of using mAb probes to assess ligand-induced conformations of troponin subunits in physiological conditions. The assays utilize relatively small amounts of protein and are free of protein modification, which may bias results. Detailed methodologies using various monoclonal antibodies (mAbs) are discussed with considerations for the optimization of assay conditions and the broader application in studies of other proteins as well as in screening of therapeutic reagents that bind a specific target site with conformational and functional effects.

Using monoclonal antibody probes in a high throughput microplate assay and localized surface plasmon resonance (LSPR) analysis of allosteric conformations of troponin subunits

Based on the concept that the specificity and affinity of antibody-antigen binding are dependent on 3-D structural fit between the antigenic epitope and the antibody variable region, site-specific monoclonal antibodies (mAbs) can be used as probes to detect the allosteric conformation and changes of a target protein. Unlike traditional methodologies such as fluorescence labeling which may alter the native state of a protein, the mAb approach can be utilized free of protein modification using relatively small amounts of the target protein under physiological conditions.

Site-Specific Detection of Tyrosine Phosphorylated CD95 Following Protein Separation by Conventional and Phospho-Protein Affinity SDS-PAGE.

Phosphorylation of two tyrosines in the death domain of CD95 is a critical mechanism in determining the receptor's choices between cell death and survival signals. Recently, site-specific monoclonal antibodies against phosphorylated tyrosines of CD95 have been generated and used to successfully detect each phosphorylated death domain tyrosine of CD95 directly and separately by immunoblotting. Here we provide detailed protocols and useful tips for a successful site-specific detection of phosphorylated death domain tyrosine of CD95 following a protein separation by sizes (conventional SDS-PAGE) and by degrees of phosphorylation (phospho-protein affinity, mobility shift SDS-PAGE).

Discrimination of rat Brunner's gland carbohydrate antigens by site-specific monoclonal antibodies

Mucus produced and secreted by gastrointestinal mucosa contains various types of mucins equipped with unique sugar chains considered to play critical roles in protecting mucous membranes; therefore, the identification and verification of mucin sugar chains is important for understanding the underlying mechanisms. In our previous work, we generated three monoclonal antibodies (mAbs), RGM22, RGM26, and RGM42, which recognize sugar chains in rat gastric mucin. Here, we immunohistochemically analyzed the rat gastrointestinal mucosa and found that the antigens recognized by RGM22 and RGM42 were expressed in the rat antrum and Brunner's glands, whereas that recognized by RGM26 was detected in the antrum, but rarely in Brunner's glands. We found that these antibodies reacted with porcine gastric mucin-derived oligosaccharides bearing a common structure: GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ1-6GalNAc-ol. Moreover, epitope analysis revealed that RGM42 and RGM22 recognized α-linked GalNAc and GalNAcα1-3Gal, respectively, on the GalNAcα1-3(Fucα1-2)Gal structure, whereas RGM26 was specific for GalNAcα1-3(Fucα1-2)Gal. These results indicate that rat Brunner's glands express specific antigens bearing GalNAcα1-3Gal that are recognized by RGM22 and RGM42. Thus, RGM22, RGM26, and RGM42 with their unique antigen specificities could be useful tools for investigation of oligosaccharide diversity among mucins.

Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies.

Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins.

Nesprins: tissue-specific expression of epsilon and other short isoforms.

Nesprin-1-giant and nesprin-2-giant regulate nuclear positioning by the interaction of their C-terminal KASH domains with nuclear membrane SUN proteins and their N-terminal calponin-homology domains with cytoskeletal actin. A number of short isoforms lacking the actin-binding domains are produced by internal promotion. We have evaluated the significance of these shorter isoforms using quantitative RT-PCR and western blotting with site-specific monoclonal antibodies. Within a complete map of nesprin isoforms, we describe two novel nesprin-2 epsilon isoforms for the first time. Epsilon isoforms are similar in size and structure to nesprin-1-alpha. Expression of nesprin isoforms was highly tissue-dependent. Nesprin-2-epsilon-1 was found in early embryonic cells, while nesprin-2-epsilon-2 was present in heart and other adult tissues, but not skeletal muscle. Some cell lines lack shorter isoforms and express only one of the two nesprin genes, suggesting that either of the giant nesprins is sufficient for basic cell functions. For the first time, localisation of endogenous nesprin away from the nuclear membrane was shown in cells where removal of the KASH domain by alternative splicing occurs. By distinguishing between degradation products and true isoforms on western blots, it was found that previously-described beta and gamma isoforms are expressed either at only low levels or with a limited tissue distribution. Two of the shortest alpha isoforms, nesprin-1-alpha-2 and nesprin-2-alpha-1, were found almost exclusively in cardiac and skeletal muscle and a highly conserved and alternatively-spliced exon, available in both nesprin genes, was always included in these tissues. These “muscle-specific” isoforms are thought to form a complex with emerin and lamin A/C at the inner nuclear membrane and mutations in all three proteins cause Emery-Dreifuss muscular dystrophy and/or inherited dilated cardiomyopathy, disorders in which only skeletal muscle and/or heart are affected.

P.5.18 Tissue-specific expression of nesprin isoforms and its relevance to muscular dystrophy and dilated cardiomyopathy

A combination of isoform-specific, quantitative RT-PCR and site-specific monoclonal antibodies has been used to produce consistent evidence at both RNA and protein levels for the principal isoform products of SYNE1 and SYNE2, the genes encoding nesprin-1 and nesprin-2 proteins. Western blots show that both genes produce three groups of isoforms that are homologous in size and structure: giant, or full-length, isoforms of about 1000 kDa, medium isoforms of about 380 kDa (nesprin-1-beta and nesprin-2-gamma) and short isoforms of about 110 kDa (nesprin-1-alpha and nesprin-2-epsilon). SYNE2 alone produces a very short isoform at 60 kDa (nesprin-2-alpha) in skeletal muscle. Although the giant forms were the dominant isoforms in all tissues, major differences between tissues in expression of shorter isoforms were observed. From SYNE1, nesprin-1-alpha was found in skeletal and cardiac muscle only. Several cell lines, including embryonic stem cells, did not produce any detectable nesprin-1 at all. From SYNE2, we observed a developmental transition from nesprin-2-epsilon-1 at very early stages (embryonic stem cells, embryonal teratocarcinoma and ovary tissue) to nesprin-2-epsilon-2 in adult and fetal tissues. Both skeletal muscle and heart have a high proportion of short isoforms compared with other tissues, but heart expresses the nesprin-2-epsilon-2 isoform where skeletal muscle produces nesprin-2-alpha-1. This study has clarified the major nesprin isoforms expressed in cells and tissues, especially in cardiac and skeletal muscle, which are the major affected tissues in neuromuscular disease. Mutations in nesprin-1 and/or nesprin-2 can cause both Emery-Dreifuss muscular dystrophy and dilated cardiomyopathy. The operative functional unit appears to be the “LINC” complex of emerin, lamin A/C, nesprins and SUN proteins, since mutations in any single component can result in a similar pathology.

Human heart failure is accompanied by altered protein kinase A subunit expression and post-translational state

β-Adrenergic receptor blockade reduces total mortality and all-cause hospitalizations in patients with heart failure (HF). Nonetheless, β-blockade does not halt disease progression, suggesting that cAMP-dependent protein kinase (PKA) signaling downstream of β-adrenergic receptor activation may persist through unique post-translational states. In this study, human myocardial tissue was used to examine the state of PKA subunits. As expected, total myosin binding protein-C phosphorylation and Ser23/24 troponin I phosphorylation significantly decreased in HF. Examination of PKA subunits demonstrated no change in type II regulatory (RIIα) or catalytic (Cα) subunit expression, although site specific RIIα (Ser96) and Cα (Thr197) phosphorylation were increased in HF. Further, the expression of type I regulatory subunit (RI) was increased in HF. Isoelectric focusing of RIα demonstrated up to three variants, consistent with reports that Ser77 and Ser83 are in vivo phosphorylation sites. Western blots with site-specific monoclonal antibodies showed increased Ser83 phosphorylation in HF. 8-fluo-cAMP binding by wild type and phosphomimic Ser77 and Ser83 mutant RIα proteins demonstrated reduced Kd for the double mutant as compared to WT RIα. Therefore, failing myocardium displays altered expression and post-translational modification of PKA subunits that may impact downstream signaling.

Detection of the dystroglycanopathy protein, fukutin, using a new panel of site-specific monoclonal antibodies

Mutations in the gene encoding fukutin protein cause Fukuyama muscular dystrophy, a severe congenital disorder that occurs mainly in Japan. A major consequence of the mutation is reduced glycosylation of alpha-dystroglycan, which is also a feature of other forms of congenital and limb-girdle muscular dystrophy. Immunodetection of endogenous fukutin in cells and tissues has been difficult and this has hampered progress in understanding fukutin function and disease pathogenesis. Using a new panel of monoclonal antibodies which bind to different defined sites on the fukutin molecule, we now show that fukutin has the predicted size for a protein without extensive glycosylation and is present at the Golgi apparatus at very low levels. These antibodies should enable more rapid future progress in understanding the molecular function of fukutin.

Hypoxia Signature of Splice Forms of Tryptophanyl-tRNA Synthetase Marks Pancreatic Cancer Cells With Distinct Metastatic Abilities

Pancreatic cancer is one of most deadly because of its aggressive growth and high metastatic ability that correlates with intratumoral hypoxia. Earlier diagnosis and prognosis marker of pancreatic cancer is not yet available. In colorectal cancer, protein biosynthesis enzyme, tryptophanyl-tRNA synthetase (TrpRS), is up-regulated in good-prognosis tumors and down-regulated in metastatic poor-prognosis tumors. Tryptophanyl-tRNA synthetase status in pancreatic cancer is unknown. To correlate metastatic ability with hypoxia and TrpRS as a possible prognostic marker, we examined mRNA and protein expression in 2 human pancreatic cancer cell lines with different metastatic abilities and TrpRS levels using our site-specific monoclonal antibodies directed to conformation-dependent epitopes on pancreatic TrpRS. Pancreatic MIAPaCa-2, Panc-1, cervical HeLa, and prostate cancer PC-3 cells were cultivated under normoxia or in hypoxic chamber. Expression of full-length TrpRS, antiangiogenic TrpRS, cyclin B1, hypoxia-inducible factor 1α, and Glut-1 was determined with reverse transcriptase-polymerase chain reaction, immunoblotting, and immunocytochemistry. We demonstrate that hypoxia regulates differentially TrpRS splice forms. Pronounced down-regulation of full-length TrpRS by hypoxia is concomitant with higher metastatic ability. Tryptophanyl-tRNA synthetase down-regulation by hypoxia may be a factor responsible for low TrpRS in tumors with high metastatic ability. Tryptophanyl-tRNA synthetase recognizability is important for pancreatic cancer prognosis and as a new target for metastasis treatment.

Antigenic characterization of a formalin-inactivated poliovirus vaccine derived from live-attenuated Sabin strains

A candidate inactivated poliovirus vaccine derived from live-attenuated Sabin strains (sIPV), which are used in the oral poliovirus vaccine (OPV), was prepared in a large-production scale. The modification of viral antigenic epitopes during the formalin inactivation process was investigated by capture ELISA assays using type-specific and antigenic site-specific monoclonal antibodies (MoAbs). The major antigenic site 1 was modified during the formalin inactivation of Sabin 1. Antigenic sites 1–3 were slightly modified during the formalin inactivation of Sabin 2 strain. Sites 1 and 3 were altered on inactivated Sabin 3 virus. These alterations were different to those shown by wild-type Saukett strain, used in conventional IPV (cIPV). It has been previously reported that type 1 sIPV showed higher immunogenicity to type 1 cIPV whereas types 2 and 3 sIPV induced lower level of immunogenicity than their cIPV counterparts. Our results suggest that the differences in epitope structure after formalin inactivation may account, at least in part, for the observed differences in immunogenicity between Sabin and wild-type inactivated poliovaccines.

Dynamics of Protein Kinase C-mediated Phosphorylation of the Complement C5a Receptor on Serine 334

Upon agonist binding, the C5a anaphylatoxin receptor (C5aR) is rapidly phosphorylated on phosphorylation sites that are located within the C-terminal domain of the receptor. Previous studies suggested that C5aR phosphorylation proceeds in a hierarchical manner with serine 334 presenting a highly accessible priming site that controls subsequent phosphorylation at other positions. To better understand the dynamics of Ser-334 phosphorylation, we generated site-specific monoclonal antibodies that specifically react with phosphoserine 334. In differentiated U937 cells, which endogenously express C5aR, stimulation with low C5a concentrations resulted in a very rapid (t((1/2)) approximately 20 s), albeit transient, receptor phosphorylation. Whole cell phosphorylation assays with specific inhibitors as well as in vitro phosphorylation assays with recombinant enzymes and peptide substrates revealed that phosphorylation of Ser-334 is regulated by protein kinase C-beta and a calyculin A-sensitive protein phosphatase. Surprisingly, at high concentrations (>10 nM) of C5a, the protein kinase C-mediated phosphorylation of Ser-334 was essentially blocked. This could be attributed to the even faster (t((1/2)) < 5 s) binding of beta-arrestin to the receptor. Analysis of C5aR Ser/Ala mutants that possess a single intact serine residue either at position 334 or at neighboring positions 327, 332, or 338 revealed functional redundancy of C-terminal phosphorylation sites since all 4 serine residues could individually support C5aR internalization and desensitization. This study is among the first to analyze in a detailed manner, using a non-mutational approach, modifications of a defined phosphorylation site in a G protein-coupled receptor and to correlate these findings with functional parameters of receptor deactivation.

Structural comparison of human C4A3 and C4B1 after proteolytic activation by C1s

The fourth component of human complement is an essential part of the classical and lectin pathways performing multifunctional roles in both host defense and immune regulation. C4 is the most polymorphic member of the complement proteins, and complete deficiency is strongly associated with autoimmune disease, especially, systemic lupus erythematosus (SLE). Of the two C4 genes C4A, but not C4B, null alleles have been implicated as important independent disease susceptibility genes occurring in more than half of SLE patients. Whether and how this deficiency contributes to the development or pathology remains unclear. We do know that activation of C4 by C1s cleaves the thioester bond, thus inducing a conformational change that exposes numerous ligand-binding sites involved in functional activity. Structural comparison, among many other tools, plays an important role in predicting function. In this report, the tertiary structures of C4A and C4B were compared using near and far-UV circular dichroism, ANS fluorescence, site-specific monoclonal antibodies and isoelectric focusing. Negligible differences in the native proteins were found. However, the activated proteins were dissimilar in secondary and tertiary structure that was accompanied by significant differences in charge distribution and surface hydrophobicity. These conformational differences, together with known acceptor preferences, have functional implications for the association between C4A null alleles and SLE.

The Hyphal and Yeast Forms ofCandida albicansBind the Complement Regulator C4b-Binding Protein

Candida albicans, an important pathogenic yeast, activates all three pathways of the complement system. To understand how this yeast evades the effects of the activated system, we have analyzed the binding of the classical pathway inhibitor C4b-binding protein (C4BP) by C. albicans. Purified native as well as recombinant C4BP bound dose dependently to the yeast and hyphal forms, as shown by multiple methods, such as confocal microscopy, flow cytometry, a novel enzyme-linked immunosorbent assay, absorption from human serum, and direct binding assays with purified proteins. A prominent binding site was identified at the tip of the germ tube, a structure that is considered important for tissue penetration and pathogenesis. The binding site in C4BP was localized to the two N-terminal complement control protein domains by using recombinant deletion constructs and site-specific monoclonal antibodies. As the alternative pathway inhibitors factor H and FHL-1 also bind to C. albicans, the binding of all three plasma proteins was compared. Simultaneous binding of the classical regulator C4BP and the alternative pathway regulator factor H was demonstrated by confocal microscopy. In addition, FHL-1 competed for binding with C4BP, suggesting that these two related complement regulators bind to the same structures on the yeast surface. The surface-attached C4BP maintains its complement regulatory activities and inactivates C4b. The surface-attached human C4BP serves multiple functions relevant for immune evasion and likely pathogenicity. It inhibits complement activation at the yeast surface and, in addition, mediates adhesion of C. albicans to host endothelial cells.

Regional Analysis of Differently Phosphorylated Tau Proteins in Brains from Patients with Alzheimer’s Disease

Neurofibrillary tangles (NFT) in Alzheimer’s disease (AD) are composed of abnormally phosphorylated tau proteins. Many phosphorylation sites have been reported in the AD brain, and NFT distribution was now roughly classified into 3 stages by Braak stage; this classification is based on pathological studies using the specific silver impregnation technique. The aim of our study was to examine the regional distribution of differently phosphorylated tau proteins with 5 site-specific monoclonal antibodies against the tau proteins, AT8, AT180, HT7, Tau2 and Tau5. We then compared our findings with those obtained from silver-stained NFT in an attempt to clarify the relationship between abnormal phosphorylation sites of the tau protein and NFT development. AT180 and AT8 labeled the highest and Tau2 the lowest density of NFT in any regions, while Tau5 and HT7 showed inconsistent distribution. In the limbic cortex, cornu ammonis, entorhinal cortex and cingulate cortex, silver-stained NFT density significantly correlated with density of NFT labeled with the 5 anti-tau antibodies, but cerebral isocortices showed heterogenous patterns of tau-positive NFT. Quantification of tau-positive regional NFT density showed that the AD-associated phosphorylation process progresses from the C-terminal to the N-terminal of the amino acid sequence, and correlation of Gallyas-stained NFT density with tau-labeled NFT density was more significant in the limbic cortices than the cerebral isocortices, which implies that stereotypical phosphorylation occurs in the limbic structures.

The effect of site-specific monoclonal antibodies directed to toxic shock syndrome toxin-1 in experimentalStaphylococcus aureusarthritis

Staphylococcus aureus produces a large number of potential virulence factors, among these the superantigen toxic shock syndrome toxin-1 (TSST-1). We have recently demonstrated that TSST-1 is involved in the pathogenesis of septic arthritis. Recent data show that the TSST-1 molecule is composed of two distinct domains, one proposed to interact with T cell receptor (TCR) and one with the MHC class II. The aim of this study was to assess if interaction between TSST-1-specific MoAbs directed to sites on the MHC and/or TCR Vbeta affects the development of experimental S. aureus-induced arthritis. For that purpose we used a panel of seven MoAbs, which were injected intraperitoneally before and after inoculation with a TSST-1-producing S. aureus strain. Administration of antibodies did not affect the development of arthritis, suggesting inefficacy of such a procedure in neutralization of exotoxin-mediated disease manifestations.

Characterization of amyloid beta protein species in the plasma, cerebrospinal fluid and brains of patients with Alzheimer's disease

Extracellular deposition of amyloid beta protein (A beta) as senile plaques and cerebral amyloid angiopathy (CAA) is one of the essential pathological characteristics of Alzheimer's disease (AD). Several A beta species with different carboxyl termini, including A beta 42 (43) and A beta 40 ending at residue 42 (43) and 40, respectively, have been identified in CAA and in senile plaque cores. Because A beta 42 (43), the major component of diffuse plaque which is the earliest pathological change in AD brains, forms insoluble amyloid fibrils more rapidly than does A beta 40, it has been hypothesized that A beta 42 (43) plays a role in amyloid seeding and A beta 40, in the elongation of amyloid fibrils on a seed of A beta 42 (43). We used enzyme-linked immunosorbent assay (ELISA) with site-specific monoclonal antibodies to differentiate A beta 42 (43) from A beta 40. First, we measured the amounts of different A beta species in plasma from patients with sporadic probable AD, age-matched patients with neurologic diseases but without dementia, and age-matched normal controls. Concentrations of A beta 1-40 and A beta 1-42 (43) in plasma did not differ significantly among the three groups. Second, CSF levels of A beta species (CSF-A beta) with different carboxy termini, i.e., A beta X-40 and A beta X-42 (43) as well as A beta 1-40 and A beta 1-42 (43), were measured in patients with AD and in age-matched controls without dementia using ELISA. Levels of both CSF-A beta X-42 (43) and A beta 1-42 (43) were significantly lower in the patients with AD that in the controls, but neither the levels of CSF-A beta X-40 nor those of CSF-A beta 1-40 differed between the two groups, which suggest that increased adsorption of A beta 42 (43) to A beta deposition in AD brains, decreased secretion of A beta 42 (43) in CSF, or increased clearance of A beta 42 (43) from CSF might explain the low levels of A beta 42 (43) in the CSF of patients with AD. Third, we measured the concentrations of various A beta species post-mortem in the cerebral cortex of patients with PS-1 mutations and beta amyloid precursor protein (APP) 717 mutation linked to familial AD or Down syndrome. The results indicate that one effect of PS-1 mutations, APP717 mutation and Down syndrome is to cause dramatic and accelerated accumulation of A beta 42 (43) in the brain as compared with sporadic AD. In particular, the increases in A beta 1-42 (43) showed a crude inverse correlation with the age of onset in each subtype of AD. Thus, quantitative studies differentiating A beta 42 (43) from A beta 40 have established the fundamental importance of A beta 42 (43) in AD.

Purification of Pregnancy Zone Protein and Its Receptor Binding Domain from Human Plasma

A new significantly improved method for purification of pregnancy zone protein (PZP), α2-macroglobulin (α2M), and the C-terminal PZP receptor binding domain is presented. Several steps in an earlier procedure have been deleted, and modifications in the gradients in the DEAE step leave most of the contaminants bound to a DEAE-Sephacel gel. This procedure makes possible the rapid, simultaneous purification of both of these closely related unstable proteins in native form from human plasma, with no thiolester cleavage or formation of tetrameric PZP. The final preparations of both α2M and PZP are pure as determined by nonreducing and reducing polyacrylamide gel electrophoresis following silver staining and no cross-contamination can be observed. The yield has been significantly improved and typically more than 500 mg PZP can be obtained from 1 liter pregnancy plasma. Furthermore, the stability of PZP at different temperatures on storage was studied. In liquid nitrogen PZP can be maintained in native dimeric form with intact thiolester for many years. The storage of native PZP with intact functional properties during and after purification is an obligatory prerequisite to elucidate the biological role of PZP. The receptor binding domain of PZP can be cleaved from the PZP–methylamine complex by papain and isolated from the other peptides by S-200 gel filtration. The cleavage site was determined and the C-terminal fragment was identified with several site-specific monoclonal antibodies against PZP.

Two Separate Domains of Laminin Promote Lung Organogenesis by Different Mechanisms of Action

Laminin is a major component of basement membranes. We previously reported that the globular region of laminin B chain(s) and the cross region of the A chain play an active role in mouse lung branching morphogenesis. In this study, basic morphogenic cell behaviors modulated by laminin were analyzed in order to elucidate how this glycoprotein promotes lung development. Cocultures of epithelial and mesenchymal cells from mouse fetal lungs were used to determine the effect of site-specific monoclonal antibodies to laminin (AL-1, AL-2, AL-3, AL-4, and AL-5) on epithelial and mesenchymal cell adhesion, proliferation, and organotypic rearrangement. We found that monoclonal antibody AL-1, directed against the cross region of the laminin A chain, inhibited epithelial and mesenchymal cell attachment and had a selective antiproliferative effect on epithelial cells. In contrast, monoclonal antibody AL-5, directed against the globular region of the B chain(s), blocked epithelial cell polarity. Immunohistochemical studies on epithelial-masenchymal cocultures exposed to monoclonal antibody AL-5 revealed the absence of laminin deposition at the epithelial-mesenchymal interface, whereas type collagen IV was present at this site. These findings suggest that each of the two laminin domains involved in lung development promotes morphogenesis by a different mechanism of action. The cross-region of the A chain mediates cell adhesion and epithelial cell proliferation, whereas the globular region of the laminin B chain(s) is critical for the process of basement membrane assembly and cell polarization. The combined effect of both laminin domains on epithelial and mesenchymal cells and on the interaction between them seems to be essential for normal lung branching morphogenesis.

Electron microscopy of antibody complexes of influenza virus haemagglutinin in the fusion pH conformation.

Activation of the membrane fusion potential of influenza haemagglutinin (HA) at endosomal pH requires changes in its structure. X-ray analysis of TBHA2, a proteolytic fragment of HA in the fusion pH conformation, indicates that at the pH of fusion the 'fusion peptide' is displaced by > 10 nm from its location in the native structure to the tip of an 11 nm triple-stranded coiled coil, and that the formation of this structure involves extensive re-folding or reorganization of HA. Here we examine the structure of TBHA2 with the electron microscope and compare it with the fusion pH structure of HA2 in virosomes, HA2 in aggregates formed at fusion pH by the soluble, bromelain-released ectodomain BHA and HA2 in liposomes with which BHA associates at fusion pH. We have oriented each HA2 preparation for comparison, using site-specific monoclonal antibodies. We conclude that the structural changes in membrane-anchored and soluble HA preparations at the pH of fusion appear to be the same; that in the absence of a target membrane, the 'fusion peptide' of HA in virosomes associates with the virosome membrane so that HA2 is membrane bound at both N- and C-termini, which implies that inversion of the re-folded HA can occur; and that the structural changes observed by X-ray analysis do not result from the proteolytic digestions used in the preparation of TBHA2.