Mapping the functional domains of human transcobalamin using monoclonal antibodies

Recombinant human transcobalamin (TC) was probed with 17 monoclonal antibodies (mAbs), using surface plasmon resonance measurements. These experiments identified five distinct epitope clusters on the surface of holo-TC. Western blot analysis of the CNBr cleavage fragments of TC allowed us to distribute the epitopes between two regions, which spanned either the second quarter of the TC sequence GQLA...TAAM(103-198) or the C-terminal peptide LEPA...LVSW(316-427). Proteolytic fragments of TC and the synthetic peptides were used to further specify the epitope map and define the functional domains of TC. Only one antibody showed some interference with cobalamin (Cbl) binding to TC, and the corresponding epitope was situated at the C-terminal stretch TQAS...QLLR(372-399). We explored the receptor-blocking effect of several mAbs and heparin to identify TC domains essential for the interaction between holo-TC and the receptor. The receptor-related epitopes were located within the TC sequence GQLA...HHSV(103-159). The putative heparin-binding site corresponded to a positively charged segment KRSN...RTVR(207-227), which also seemed to be necessary for receptor binding. We conclude that conformational changes in TC upon Cbl binding are accompanied by the convergence of multiple domains, and only the assembled conformation of the protein (i.e. holo-TC) has high affinity for the receptor.     

Localization of three distinct heparin-binding domains of laminin by monoclonal antibodies

Monoclonal antibodies were utilized to localize novel heparin-binding domains of laminin. A solid-phase radioligand binding assay was designed such that [3H] heparin bound to laminin in a time- and concentration-dependent manner. Tritiated heparin binding to laminin was saturable and specific as determined by competition with unlabeled heparin, dextran sulfate, and dermatan sulfate. By Scatchard analysis, two distinct dissociation constants were calculated (Kd = 50 and 130 nM), suggesting the presence of at least two binding sites for heparin on laminin. Tritiated heparin bound to thrombin-resistant (600 kDa) and chymotrypsin-resistant (440 kDa) laminin fragments, both known to lack the terminal globular domain of the long arm. Sodium dodecyl sulfate-polyacrylamide gels of chymotrypsin- and thermolysin-digested laminin chromatographed on a heparin-Sepharose column showed multiple proteolytic fragments binding to the column. Monoclonal antibodies generated against laminin were tested for their ability to inhibit [3H]heparin binding to laminin. Four monoclonal antibodies significantly inhibited the binding of [3H]heparin to laminin in the range of 15-21% inhibition. Laminin-monoclonal antibody interactions examined by electron microscopy showed that one antibody reacted at the terminal globular domain of the long arm, domain Hep-1, while epitopes for two of these monoclonal antibodies were located on the lateral arms of laminin, domain Hep-2, and the fourth monoclonal antibody bound below the cross-region of laminin, domain Hep-3. When two monoclonal antibodies recognizing distinctly different regions of laminin were added concomitantly, the inhibition of [3H]heparin binding to laminin increased almost 2-fold. These results suggest that at least two novel heparin-binding domains of laminin may be located in domains distinct from the terminal globular domain of the long arm.     

Ultrastructural localization of human kidney antigens using monoclonal antibodies.

A highly sensitive method of ultrastructural-immunoperoxidase staining was developed for use with monoclonal antibodies which have been raised in this laboratory to a variety of antigens of the human kidney. Because of the susceptibility of the antigens to fixation and processing, a four layer, pre-embedding method of staining was used. Results confirmed and clarified previously reported light microscopy results, indicating that an antigen recognized by the PHM5 antibody was found on the podocyte cell membrane within the glomerulus and was not present within the glomerular basement membrane. The antigen was also present on the extraglomerular endothelial cell membrane. The study also demonstrated the presence of an antigen specific to endothelial cells throughout the renal cortex, and gave further insight into the precise localization of glomerular basement membrane components including fibronectin. The method of staining is now being used together with detailed ultrastructural studies to identify the cells produced from isolated glomeruli in tissue culture.     

Mapping of functional domains of human high molecular weight and low molecular weight kininogens using murine monoclonal antibodies

Thirty-four monoclonal antibodies directed against human high molecular weight (HMW) and low molecular weight (LMW) kininogens and their derivatives were obtained, and the specificities of the antibodies were assayed by enzyme-linked immunosorbent assay (ELISA). By use of HMW kininogen, kinin-free HMW kininogen, kinin-free and fragment 1.2 (fr 1.2) free HMW kininogen, fr 1.2-light chain of HMW kininogen, LMW kininogen, kinin-free LMW kininogen, heavy chain of LMW kininogen, and light chain of LMW kininogen, the monoclonal antibodies were characterized and classified into four groups: (A) 20 monoclonal antibodies reacting with only the heavy chain, a common region of HMW and LMW kininogens; each of these monoclonal antibodies possessed the specificity to domain 1 (2 monoclonal antibodies), domain 2 (2 monoclonal antibodies), domain 3 (7 monoclonal antibodies), and both domains 2 and 3 (7 monoclonal antibodies) of the heavy chain; (B) 7 monoclonal antibodies reacting with fr 1.2, a unique histidine-rich region; (C) 5 monoclonal antibodies reacting with the light chain of HMW kininogen; (D) 2 monoclonal antibodies reacting with the light chain of LMW kininogen. Two monoclonal antibodies in the first group (group A), designated HKG H7 and H12, effectively suppressed the thiol proteinase inhibitor activity of HMW kininogen to papain and calpains and of LMW kininogen to papain, but the others did not affect it. Further, all the monoclonal antibodies which recognized the fr 1.2 or light chain of HMW kininogen (groups B and C) suppressed the clotting activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mapping two functional domains of clathrin light chains with monoclonal antibodies

The two forms of clathrin light chains (LCA and LCB) or clathrin-associated proteins (CAP1 and CAP2) have presented an immunochemical paradox. Biochemically similar, both possess two known functional parameters: binding the clathrin heavy chain and mediating the action of an uncoating ATPase. All previously reported anti-CAP mAbs, however, react specifically with only CAP1 (Brodsky, F. M., 1985, J. Cell Biol., 101:2047-2054; Kirchhausen, T., S. C. Harrison, P. Parham, and F. M. Brodsky, 1983, Proc. Natl. Acad. Sci. USA, 80:2481-2485). Four new anti-CAP mAbs are reported here: two, C-7H12 and C-6C1, react with both forms; two others, C-10B2 and C-4E5, react only with the lower form. Sandwich ELISAs indicated that C-10B2, C-4E5, C-6C1, and C-7H12 react with distinct epitopes. Monoclonal antibodies C-10B2 and C-4E5 immunoprecipitate clathrin-coated vesicles (CCVs) and react with CAP2 epitopes accessible to chymotrypsin on the vesicle. These mAbs inhibit phosphorylation of CAP2 by endogenous CCV casein kinase II. In contrast, C-6C1 and C-7H12 react with epitopes that are relatively insensitive to chymotrypsin. CAP peptide fragments containing these epitopes remain bound to reassembled cages or CCVs after digestion. Immunoprecipitation and ELISAs demonstrate that C-7H12 and C-6C1 react with unbound CAPs but not with CAPs bound to triskelions or CCVs. The data indicate that the CAPs consist of at least two discernible structural domains: a nonconserved, accessible domain that is relevant to the phosphorylation of CAP2 and a conserved, inaccessible domain that mediates the binding of CAPs to CCVs.     

Mapping of distinct structural domains on microtubule-associated protein 2 by monoclonal antibodies

Monoclonal antibodies against microtubule-associated protein 2 (MAP2) were prepared and their specificity was verified by visualization of the antigens using the antibody overlay technique and by radioimmunoassay. MAP2 was cleaved by alpha-chymotrypsin to generate a series of high-molecular-mass fragments ranging between 270 and 140 kDa. The precursor-product relationship of these fragments was suggested from the rate of their appearance and from the analysis of the tryptic peptide map of each fragment. A group of monoclonal antibodies was found to react predominantly with the intact 270-kDa MAP2 molecule and a fragment having a mass of 240 kDa and to some extent with a 215-kDa fragment. Another group of monoclonal antibodies reacted with an antigenic determinant which was located on the 270-kDa molecule as well as on fragments as small as 140 kDa. None of the two groups of monoclonal antibodies reacted with the microtubule-binding domain of MAP2. These results suggest that one group of antibodies reacts with sites located at or dependent upon a terminal 60-kDa domain(s) distal to the microtubule-binding site of MAP2. The second group of antibodies, which can still bind to smaller proteolytic products, appear to be associated with the central region of the MAP2 molecule. Indirect immunofluorescence experiments with the antibody preparations indicated that at least some of the antigenic determinants are exposed when MAP2 is associated with microtubules in the cell body and neurite outgrowths of differentiated rat brain neuroblastoma B104 cells.     

Mapping of biologically relevant sites on human IL-1 beta using monoclonal antibodies

mAb have been raised that recognize human IL-1 beta. Using overlapping peptide fragments expressed in yeast and bacteria, we have mapped the regions of the protein to which these antibodies bind. To assess the relevance of the different regions of IL-1 beta for the expression of its biologic activity, the ability of the antibodies to block IL-1 activity was assayed. Antibodies recognizing the regions 133-148 and 251-269 of human IL-1 beta could inhibit the activity of IL-1 beta, but not of IL-1 alpha, in two different biologic assays, the murine thymocyte proliferation and PGE2 release from human fibroblasts. Conversely, antibodies that recognize the region 218-243 have only a moderate inhibitory effect on the IL-1 beta biologic activity in both assays. Finally, an antibody mapping to the region 148-192 did not inhibit IL-1 beta activity either on thymocytes or on fibroblasts. It is suggested that IL-1 beta-induced cell activation involves different regions of the protein and that both N-terminal and C-terminal fragments are involved in the correct functioning of the IL-1 beta molecule.     

Characterization of functionally important domains in human vitamin K-dependent protein S using monoclonal antibodies.

Vitamin K-dependent protein S is an anticoagulant plasma protein functioning as a cofactor to activated protein C in the degradation of coagulation factors Va and VIIIa. To determine which regions in protein S are important for its cofactor activity, we have raised and characterized a large panel of monoclonal antibodies against human protein S. Several of the antibodies were directed against Ca2(+)-dependent epitopes, and they were found to be located either in the domain containing gamma-carboxyglutamic acid (Gla), the thrombin-sensitive region, or in the first epidermal growth factor (EGF)-like domain. The first two types of epitopes were exposed at approximately 1 mM Ca2+, whereas the epitope(s) in the EGF-like domains required less than 1 microM Ca2+, suggesting the presence of one or more high affinity Ca2(+)-binding site(s). The antibodies, as well as their Fab' fragments, against all three types of Ca2(+)-dependent epitopes efficiently inhibited the activated protein C cofactor function of protein S, but through different mechanisms. The antibodies against the Gla domain exerted their effects through inhibition of protein S binding to negatively charged phospholipid. Fab'-fragments of antibodies against the thrombin-sensitive region and the first EGF-like domain were the most potent inhibitors of the activated protein C cofactor function but did not inhibit phospholipid binding of protein S. In conclusion, we have identified the domains in protein S that are important for the activated protein C cofactor activity. The Gla domain is instrumental in the binding of protein S to phospholipid, whereas the thrombin-sensitive region and the first EGF-like domain may be directly involved in protein-protein interactions on the phospholipid surface.     

Schwannoma cell-derived inhibitor of the neurite-promoting activity of laminin

During the purification of laminin-proteoglycan complexes from rat RN22 Schwannoma cell-conditioned medium, a laminin-rich fraction was obtained which lacked neurite-promoting activity. Since laminin from several sources is known to have potent neurite-promoting activity, this result suggested that either this laminin was inactive or its activity was somehow masked by associated molecule(s). The latter possibility was supported by the demonstration that the inactive laminin-containing fraction inhibited active laminin-containing fractions. This inhibitory activity was partially purified by using ion exchange chromatography and isopycnic centrifugation. The purified material contained proteoglycan based on its high affinity for cationic resin, high buoyant density, large heterodisperse appearance on electrophoretic gels, ability to label with inorganic sulfate, sensitivity to trypsin and glycosaminoglycan lyases, and heat stability. A quantitative in vitro bioassay was used to monitor the inhibitor after treatments aimed at defining its activity. The isolated Schwannoma-derived inhibitor (a) inhibits the neurite-promoting activity of purified rat, mouse, and human laminin; (b) is active whether presented to laminin in solution or after either the inhibitor or laminin is first bound to the culture substratum; (c) does not act by displacing laminin from the substratum; (d) can be prevented from binding to neurite-promoting laminin substrates by polyclonal and monoclonal anti-laminin or polyclonal anti-entactin antibodies; and (e) is abolished by proteases or glycosaminoglycan lyases but not by heat. The above results suggest that the neurite-promoting activity of laminin is subject to regulation through association with a proteoglycan and entactin.     

Identification of laminin domains involved in branching morphogenesis: effects of anti-laminin monoclonal antibodies on mouse embryonic lung development

We recently found that polyclonal antibodies to laminin, a basement membrane-related glycoprotein, inhibited murine lung morphogenesis when added to organ cultures of mouse embryonic lung. Using a series of monoclonal anti-laminin antibodies with previously characterized subunit specificity (termed AL-1, AL-2, AL-3, AL-4, and AL-5), the deposition and functional involvement of different laminin domains in the developing lung were investigated. By immunohistochemistry the antibodies' reactivity was largely localized to the basement membrane, but was also present diffusely in the extracellular matrix throughout the mesenchyme. Organ cultures of lung explants from Day 12 embryos were cultured for 3 days in the presence of 50-100 micrograms/ml of each antibody or in the presence of the same concentration of immunoglobulins G and M, laminin-neutralized antibody, or medium alone. Cultures were monitored by phase-contrast microscopy, light microscopy, and immunofluorescence. Although all antibodies penetrated the tissues in culture, only two of them inhibited branching activity. These two antibodies were AL-1, which binds on or near the cross region of laminin, and AL-5, which binds to the lateral short arms at the globular end regions of the B chain of laminin. Inhibition of branching with these two antibodies was dose-dependent and statistically significant for the two concentrations used. AL-2, AL-3, AL-4, laminin-neutralized antibodies and control immunoglobulins did not alter lung morphogenesis. The two domains of laminin that promote lung branching morphogenesis have been reported by others to promote the attachment of a variety of cells and/or bind heparin. These domains of laminin may promote branching morphogenesis by facilitating cell attachment and, consequently, cell proliferation.     

Localization of the site of human transferrin interacting with cellular receptors using monoclonal antibodies]

Antigenic determinants of the human transferrin molecule on the sublobe and lobe levels were localized for 7 monoclonal antibodies. Antibodies used have different effects on the interaction of the transferrin with its receptor. It was concluded that transferrin-receptor recognition was determined by NH2-lobe, the N2-sublobe playing major part. Dimerization of the transferrin molecules in solution was detected. Using the panel of monoclonal antibodies it was shown that dimerization accomplished by means of the COOH-lobes of transferrin molecules, the sites of interaction of the NH2-lobe with receptor being exposed. A model of the transferrin - receptor complex is proposed.     

Topographical localization of distinct antigenic domains of Toxoplasma gondii with the aid of monoclonal antibodies

In the present study, a panel of six individual hybridoma derived antibodies produced against the BK strain of Toxoplasma gondii were evaluated for their reactivity in an immunofluorescence test. Each of the six monoclonal antibodies demonstrated a unique pattern of fluorescence localized to distinctive regions on the toxoplasmas. Although all of the six detected antigenic determinants which were shared by at least four different T. gondii strains, monoclonal antibody TG-E4A17 has disclosed hitherto unrecognized population differences among the BK or PH strain parasites. The fact that some of the antigenic molecules are restricted to distinct regions of the toxoplasmas may have implications in the infections process/immune response.     

Mapping surface structures of the human insulin receptor with monoclonal antibodies: localization of main immunogenic regions to the receptor kinase domain

A panel of 37 monoclonal antibodies to the human insulin receptor has been used to characterize the receptor's major antigenic regions and their relationship to receptor functions. Three antibodies recognized extracellular surface structures, including the insulin binding site and a region not associated with insulin binding. The remaining 34 monoclonal antibodies were directed against the cytoplasmic domain of the receptor beta subunit. Competitive binding studies demonstrated that four antigenic regions (beta 1, beta 2, beta 3, and beta 4) are found on this domain. Sixteen of the antibodies were found to be directed against beta 1, nine against beta 2, seven against beta 3, and two against beta 4. Antibodies to all four regions inhibited the receptor-associated protein kinase activity to some extent, although antibodies directed against the beta 2 region completely inhibited the kinase activity of the receptor both in the autophosphorylation reaction and in the phosphorylation of an exogenous substrate, histone. Antibodies to the beta 2 region also did not recognize autophosphorylated receptor. In addition, antibodies to this same region recognized the receptor for insulin-like growth factor I (IGF-I) as well as the insulin receptor. In contrast, antibodies to other cytoplasmic regions did not recognize the IGF-I receptor as well as the insulin receptor. These results indicate that the major immunogenic regions of the insulin receptor are located on the cytoplasmic domain of the receptor beta subunit and are associated with the tyrosine-specific kinase activity of the receptor. In addition, these results suggest that a portion of the insulin receptor is highly homologous to that of the IGF-I receptor.     

Characterization of the glucagon receptor and its functional domains using monoclonal antibodies

Four monoclonal antibodies, designated 4H11, 6E10, 2C5, and 3E9 were prepared against partially purified rat hepatic glucagon receptor. These antibodies were characterized by their ability to recognize the glucagon receptor in target tissues using immunoblot and immunoprecipitation procedures. The antibodies recognized a 62-kDa receptor band in rat liver, kidney, and adipose tissue but not in lung, adrenals, and erythrocytes, indicating a high degree of specificity. These antibodies recognize different antigenic determinants; the 6E10 and 2C5 bind protein epitopes, while 4H11 and 3E9 bind carbohydrate epitopes. Furthermore, proteolytic mapping of the glucagon receptor established that monoclonal antibodies 6E10 and 2C5 recognize different domains of the receptor molecule. These antibodies were used to study the immunochemical similarities among the receptors from different species and to assess the topological location of the ligand-binding site. By combining the techniques of affinity cross-linking, proteolytic mapping, and antibody binding, we have identified the location of the glucagon-binding site near to the COOH-terminal domain of the receptor.     

Mapping of antigenic determinants of human apolipoprotein B using monoclonal antibodies against low density lipoproteins

The reactivity of a series of monoclonal antibodies directed against human low density lipoproteins (LDL) has been tested with hepatic and intestinal apolipoprotein B (apo-B) termed B-100 and B-48, respectively (Kane, J. P., Hardman, D. A., and Paulus, H. E. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2465-2469). Whereas those antibodies that have been previously shown to recognize determinants close to the LDL receptor recognition site reacted only with B-100, two antibodies specific for other regions of apo-B reacted with both B-100 and B-48. Therefore, it is probable that sequence homologies exist between the two proteins and it must be considered that all or parts of the B-48 sequence may be contained within that of B-100. The specificity of the reaction of these antibodies with proteins designated B-74 and B-26 supports the concept that they represent complementary fragments of B-100. The present results have been incorporated in a theoretical map of the antigenic determinants recognized by these antibodies on the LDL apo-B.     

Mapping of phosphorylation-dependent anti-tau monoclonal antibodies in immunoblots using human tau-constructs synthesized by native chemical ligation

In Alzheimer’s disease (AD) neurofibrillary tangles (NFT) are formed by hyperphosphorylated microtubule-associated tau protein. It is still a matter of controversy which phosphorylation sites are AD-specific and how these might be linked to the cause or progress of the disease. Whereas most research projects in this field rely on phosphorylation-dependent tau-specific monoclonal antibodies (mAbs), the phosphorylation patterns recognized by these mAbs are often not characterized in detail. Therefore, we synthesized unphosphorylated, two monophosphorylated (pThr231, pSer235), and the bisphosphorylated (pThr231 + pSer235) tau226-240 peptides. The phosphopeptides were ligated via an N-terminal cysteine to the thioester-activated C-terminus of human aldo/keto reductase AKR1A1. After purification by preparative gel electrophoresis, the ligation products were analyzed by Western blotting and probed with phosphorylation-dependent anti-tau mAbs HPT-101, HPT-103, HPT-104, and HPT-110. The obtained specificities were very similar to the data obtained by ELISA, showing that ELISA-based epitope mapping studies are also valid for immunoblot analyses.     

Analysis of the human immune repertoire using human monoclonal antibodies

The investigations of the human immune response to cancer and other diseases have been hampered by the difficulty in determining the specificity of low-titered antibodies in serum, and the inability to define the specificity of individual lymphocytes. In order to study these issues, we developed the hybridoma technology so that human monoclonal antibodies (hM Ab) could be reliably and reproducibly obtained. Using a variety of fusion partners of both mouse and human origin, a large number of human immunoglobulin-secreting hybrids have been generated. We have found that 5 to 10% of the hybridomas produced secrete hM Ab reactive with antigens (Ag) expressed by human cells. Specificity analysis and cellular localization studies of the Ag have been performed for a large number of these hM Ab, and several general points have emerged from our study: (A) A significant proportion of the evaluable B-cell repertoire is directed to the production of antibodies reactive with Ags expressed by human cells. (B) The great majority of these Ags have an intracellular location, and are broadly distributed, being expressed by both normal and malignant cells. (C) Intracellular and cell surface differentiation Ags and other Ags with restricted distribution have been defined by hM Ab, including a series of cell surface and intracellular Ags not detected on normal cells. (D) The relationship of these findings to cancer is unclear as hM Ab showing distinctive distributions have been generated from the lymphocytes of normal individuals as well as tumor-bearing patients. (E) hM Ab with distributions distinct from any known mouse monoclonal antibodies (mM Ab) have been obtained.(ABSTRACT TRUNCATED AT 250 WORDS)