Degradation of fibronectin by human leukocyte elastase. Release of biologically active fragments

We have identified the biological activity of three polypeptides released by limited proteolysis of human plasma fibronectin by leukocyte elastase. A Mr = 140,000 peptide contains cell-spreading activity; a Mr = 60,000 peptide mediates binding to denatured collagen (gelatin), and a Mr = 29,000 peptide contains glutaminyl residues responsible for the transglutaminase (blood coagulation factor XIIIa)-catalyzed incorporation of amines. More extensive proteolysis yielded numerous peptides, including a Mr = 40,000 peptide derived from the Mr = 60,000 peptide which retains gelatin-binding activity. Quantification of the gelatin-binding peptides is consistent with two binding sites per dimeric fibronectin molecule of Mr = 440,000. Both Mr = 60,000 and 40,000 gelatin-binding peptides were enriched with half-cystine residues, containing 28 and 25, respectively, but devoid of cysteine. This, coupled with the electrophoretic behavior of both peptides, was consistent with the presence of intramolecular disulfide bonds in the gelatin-binding domain. Intact fibronectin contains 1 free cysteine residue/monomer, as recently described. This cysteine reacts with 5,5'-dithiobis(2-nitrobenzoic acid) very slowly under nondenaturing conditions but rapidly when fibronectin is denatured. The free cysteine is located in the Mr = 140,000 peptide. While the Mr = 40,000 and 60,000 gelatin-binding peptides bind to gelatin with an affinity about 30-fold and 5-fold less than intact fibronectin (based on a monomeric fibronectin Mr = 220,000), neither gelatin-binding peptide supports spreading of fibronectin-deficient test cells on gelatin or tissue culture plastic substrates. The purified Mr = 140,000 peptide supported cell spreading on plastic, retaining about one-half of the spreading activity of intact fibronectin on a weight basis. These data confirm recent results, suggesting multiple, protease- resistant domains with discrete biological functions within fibronectin. Our results, together with established data, suggest a model for the location of the transglutaminase-reactive glutaminyl residues, gelatin binding, and cell-adhesive domains in fibronectin. The release of univalent, biologically active fibronectin fragments by elastase, a major physiologically released inflammatory protease of human leukocytes, suggests a new potential mechanism for alteration of cell connective tissue interactions at sites of inflammation in vivo.     

Interdomain cleavage of plasma fibronectin by zinc-metalloproteinase from Serratia marcescens

Limited proteolysis of porcine plasma fibronectin by the 56 kDa proteinase (56K proteinase) (EC 3.4.24.4) from Serratia marcescens released six polypeptides: a 27 kDa peptide, the heparin-binding domain which comprises the NH2-terminal end; a 50 kDa peptide, a mid-molecule that mediates binding to gelatin or collagen; a 160 kDa peptide, that contained the heparin-binding domain with cell-spreading activity; and a 140 and a 20 kDa peptide which released from the 160 kDa peptide. Each fragment was purified and characterized by its chemical and biological properties, and it was found that they were respectively different domains. Both the 160 and the 140 kDa peptide contained one cysteine per mole of peptide. The 160 kDa peptides were connected by a 6 kDa peptide, which was present at the COOH-terminal end of the molecule and was biologically inactive. Only 6 kDa peptide contained a disulfide bond and produced 3 kDa peptide after reduction, whereas other fragments did not change with or without reduction on SDS-polyacrylamide gel electrophoresis. NH2-terminal sequence analyses of the released peptides showed that the 56K proteinase cleaved the fibronectin between the Arg-Thr (located at two different sites), Leu-Ser and Gln-Glu bonds. Out of 118 Arg residues, there are nine sequences containing Arg-Thr, and two of them near or at an interdomain location (at Arg 259 and 2239) were cleaved. Out of 124 Leu residues, there are 11 Leu-Ser sequences and only one, at 687, was cleaved. The above fragments with functional domain activity could be aligned according to the previously reported amino-acid sequence of human or bovine plasma fibronectin. The treatment of fibroblast cells by the 56K proteinase resulted in loss of morphological integrity and extracellular matrix.     

Biosynthesis of two subunits of type IV procollagen and of other basement membrane proteins by a human tumor cell line

The major collagenous component secreted into the medium of cultured HT-1080 tumor cells was identified as type IV procollagen by specific antibodies and characteristic ratios of incorporated labeled 3-hydroxyproline and 4-hydroxyproline. The disulfide-bonded molecules consisted of two subunits, pro-alpha 1(IV) and pro-alpha 2(IV) chains with apparent molecular weights of 180 000 and 165 000. No conversion of the procollagen to collagen or to procollagen intermediates was detected in the cell cultures. The two subunits apparently represent different gene products, since enzymatic digestion of the separated chains produced quite different peptide maps. Pepsin degraded native type IV procollagen successively into several fragments, some still disulfide-linked, giving rise to a complex set of polypeptide chains (Mr = 30 000-140 000). This agrees with similar diverse patterns produced by pepsin from authentic type IV collagens. The ratio between the pro-alpha 1(IV) and pro-alpha 2(IV) chains varied in several experiments between 1.3 and 1.8, suggesting that the two chains belong to different triple-helical molecules. The cells also produced distinct amounts of fibronectin (subunit Mr = 230 000) and of the basement membrane glycoprotein laminin. The latter showed three subunits with Mr = 220 000, 210 000, and 400 000. A further disulfide-bonded, non-collagenous polypeptide (Mr = 160 000) was detected but not yet identified. Immunofluorescence demonstrated these proteins within the cells but not in a pericellular matrix. The production of basement membrane components by HT-1080 cells and lack of interstitial collagens disagree with the original classification of the cell line as a fibrosarcoma.     

Domain structure of human plasma and cellular fibronectin. Use of a monoclonal antibody and heparin affinity to identify three different subunit chains

The domain structure of human plasma fibronectin was investigated by using heparin-binding and antibody reactivity of fibronectin and its proteolytically derived fragments. Digestion of human plasma fibronectin with a combination of trypsin and cathepsin D produced six major fragments. Affinity chromatography showed that one fragment (Mr 45 000) binds to gelatin and three fragments (Mr 31 000, 36 000, and 61 000) bind to heparin. The 31K fragment corresponds to NH2-terminal fragments isolated from other species. The 36K and 61K fragments are derived from a region near the C-terminus of the molecule and appear to be structurally related as demonstrated by two-dimensional peptide maps. A protease-sensitive fragment (Mr 137 000), which binds neither gelatin nor heparin but which has been shown previously to be chemotactic for cells [Postlethwaite, A. E., Keski-Oja, J., Balian, G., & Kang, A. H. (1981) J. Exp. Med. 153, 494-499], separates the NH2-terminal heparin- and gelatin-binding fragments from the C-terminal 36K and 61K heparin-binding fragments. A monoclonal antibody to fibronectin that recognized the 61K heparin-binding fragment was used to isolate a sixth fragment (Mr 34 000) that did not bind to heparin or gelatin and that represents a difference between the 61K and 36K heparin-binding fragments. Cathepsin D digestion produced an 83K heparin-binding, monoclonal antibody reactive fragment that contains the interchain disulfide bond(s) linking the two fibronectin chains at their C-termini. The data indicate that plasma fibronectin is a heterodimeric molecule consisting of two very similar but not identical chains (A and B). In contrast, enzymatic digestion of cellular fibronectin produced a 50K heparin-binding fragment lacking monoclonal antibody reactivity which suggests that the cellular fibronectin subunit is similar to the plasma A chain in enzyme susceptibility but contains a larger heparin-binding domain. A model relating the differences in the three fibronectin polypeptides to differences in published cDNA sequences is presented.     

Identification of a fibronectin receptor specific for rat liver endothelial cells

Antibodies raised against the fibronectin receptor of rat hepatocytes recognized one protein (Mr 120 and 135 kDa for unreduced and reduced samples, respectively) in immunoblotting of solubilized rat liver endothelial cells (LEC). The antibodies specifically precipitated a 200-kDa protein together with the 135-kDa component from 125I-labeled LEC. Spreading of LEC on fibronectin, but not on laminin or collagen, was inhibited by monovalent Fab fragments of the antibodies, implicating that the 135/200-kDa complex is a specific fibronectin receptor. The results indicate that LEC, hepatocytes, and fibroblasts of rat carry different fibronectin receptors, suggesting that the interaction of fibronectin with these cells may have different functional roles.     

Structure and expression of Fc gamma receptors on mouse suppressor T cell hybridomas

Antigen-specific and idiotype-specific mouse suppressor T cell hybridomas were analyzed for the presence and specificity of Fc gamma receptors (Fc gamma R) by EA rosetting and by flow microfluorometry with the use of monoclonal antibodies. We found that four hybridomas expressed Fc gamma R specific for IgG1 and IgG2b, one of which became Fc gamma R- during prolonged culture. Four other hybridomas and the fusion parent, BW5147, consistently lacked Fc gamma R. The 125I-labeled Fc gamma R were isolated from surface radioiodinated hybridoma cells solubilized with 1% Nonidet P-40, were purified by using single or repetitive chromatography on mouse IgG-Sepharose columns, and were analyzed by SDS-PAGE. An 125I-labeled 56,000 to 61,000 Mr macromolecule was isolated from each of the Fc gamma R+ hybridomas, but from none of the Fc gamma R- hybridomas nor from BW5147 cells. This macromolecule rebound to insolubilized mouse IgG1, IgG2b, and human Fc fragments, but not to insolubilized mouse IgG2a, IgG3, or IgA or human F(ab')2 fragments, consistent with the specificity observed for Fc gamma R on intact hybridoma cells. The mouse suppressor T cell Fc gamma R differs in size and specificity from mouse B cell Fc gamma R. A 70,000 Mr protein expressed on all hybridomas and on BW5147 cells was radiolabeled and, despite preclearing with ovalbumin-Sepharose, bound to the mouse IgG-Sepharose columns, presumably due to mouse antibodies to gp-70. This macromolecule was completely and specifically removed by using goat antiserum to gp-70.     

Monoclonal antibodies to Chlamydia psittaci: their isolation, characteristics and use]

A panel of 22 hybridomas producing monoclonal antibodies (McAb) to C. psittaci was obtained. 15 hybridomas produced IgG1 antibodies, 4 hybridomas produced IgM antibodies and 3 hybridomas produced IgG2b, IgG3 or IgA antibodies. IgG1 antibodies and 2 IgM antibodies did not bind complement in the complement fixation test. All McAb were reactive in the enzyme immunoassay and the indirect immunofluorescence test and did not precipitate specific antigens. Peroxidase conjugates on the basis of McAb effectively detected Chlamydia antigen, prepared from the crude suspension of chick embryo yolk sack infected with different strains of C. psittaci and C. trachomatis, in different modifications of EIA.     

Evidence for preferential proteolytic cleavage of one of the two fibronectin subunits and for immunological localization of a site distinguishing them

Purified plasma fibronectin was digested sequentially by thrombin and cathepsin G or by cathepsin G alone and the degradation products and their gelatin-binding and heparin-binding fractions were analyzed in NaDodSO4-polyacrylamide gel electrophoresis followed by immunoblotting with a defined monoclonal anti-fibronectin antibody. In early cathepsin G digests, several gelatin-binding fragments were detected: a few large (Mr greater than or equal to 150 000) polypeptides and fragments of Mr = 85 000, 72 000, 64 000 and 40 000. The 85 000-Mr and 64 000-Mr fragments appeared as closely spaced doublets and reacted with the antibody while the 72 000-Mr and 40 000-Mr fragments did not. Therefore the 64 000-Mr fragments are likely to be derived from the 85 000-Mr fragments. Three large fragments that bound to heparin, but not to gelatin were detected: Mr = 145 000, 135 000 and 120 000. Of these only the 135 000-Mr peptide reacted with the antibody. When fibronectin was digested with thrombin, polypeptides of Mr = 180 000-200 000 and a 30 000-Mr NH2-terminal fragment were produced. Cathepsin G added to this mixture further cleaved the fragments to a digestion pattern resembling that obtained from intact fibronectin except that the 85 000-Mr and 64 000-Mr fragments appeared as single bands and the amount of the 72 000-Mr fragment was reduced. The results suggest that thrombin cleaves the 30 000-Mr fragment preferentially from the NH2-terminal end of one of the two subunits of fibronectin and that the 85 000-Mr, 72 000-Mr and 64 000-Mr fragments obtained by the additional cathepsin G digestion were derived from the other chain. The results are consistent with the model that the antigenic determinant resides 72 000-85 000 Da from the NH2-terminus and is cleaved by cathepsin G alternatively at one of its sides. Thus, the components of the 85 000-Mr and 64 000-Mr doublets are derived from different subunits and the region located by the antibody may be responsible for the difference in their migration in the polyacrylamide gel.     

Demonstration of a common antigenic site on endomembrane proteins of Phaseolus vulgaris by a rat monoclonal antibody

Five hybrid myeloma cell lines that secrete antibodies to plant endomembrane-bound proteins have been prepared using rat myelomas and spleen cells from rats immunized against intact endoplasmic-reticulum and Golgi-membrane preparations from Phaseolus vulgaris. Four of these lines produced antibodies which all showed identical binding patterns in Western blots, recognising polypeptides of Mr 35 000, 58 000, 70 000, 91 000 and possibly 117 000 common to both membrane types, while the antibody produced by the fifth line bound to a polypeptide of Mr 57 000. This binding pattern persisted for the antibody produced by all positive clones derived by extensive subcloning of hybridomas 2B3 and 2C3 even with subsequent growth, so these polypeptides, therefore, probably have a common antigenic site. The antibody tested from the hybridoma 2C3 and two subsequent subclones inhibited the arabinosyl transferase involved in the synthesis of arabinan, a component of the primary cell-wall matrix, so that one of these polypeptides probably represents the enzyme. Comparison of the patterns of the changes in enzyme activity with the levels of each individual polypeptide in cells induced to divide and undergo primary growth tentatively identifies the 70 000-Mr polypeptide as the arabinan synthase. Interpretation of this and previous data indicates that the induction of this enzyme activity by plant growth regulators involves de novo synthesis of the protein.Abbreviations PBS phosphate-buffered saline - Ig immuno-globulin - SDS sodium dodecyl sulfate     

Analysis of T and B cell epitopes of the Schistosoma mansoni P28 antigen in the rat model by using synthetic peptides

The Schistosoma mansoni P28 molecule is an Ag inducing protective immunity in various experimental models. Three synthetic peptides, derived from the primary sequence of the recombinant P28 and comprising amino acids 24-43, 115-131, and 140-153, respectively, were synthesized according to their hydrophilicity, mobility, and accessibility profiles. The presence of B and T lymphocyte epitopes in these peptides has been examined in the rat model. The results showed that the 24-43 and the 115-131 peptides contained major epitopes for IgG but not for IgE. Moreover, the 24-43 peptide-specific IgG produced after injecting either the recombinant P28 Ag or the 24-43 peptide coupled to tetanus toxoid was essentially of the IgG2a subclass and to a lesser extent of the IgG1 subclass, whereas no IgG2c was detected. These 24-43 peptide-specific antibodies were cytotoxic in vitro for schistosomula in the presence of eosinophils as effector cells. The 24-43 and the 140-153 peptides contained major targets of T lymphocytes specific for the recombinant P28 Ag. T cell lines specific for the 24-43 peptide have been prepared. These cells proliferated in vitro when stimulated with various S. mansoni crude antigenic preparations or with the recombinant P28 Ag. Moreover, their passive transfer to rats immunized with the P28 Ag led to a significant increase in specific IgE without modifying the IgG response.     

Monoclonal antibodies to the rat liver glucocorticoid receptor.

Monoclonal antibodies against the 90 000 mol. wt. form of the activated rat liver glucocorticoid receptor were generated from mice immunized with a partially purified receptor preparation. The screening assay was based on the precipitation of liver cytosol, labelled with [3H]triamcinolone acetonide, with monoclonal antibodies bound to immobilized rabbit anti-mouse IgG. Out of 102 hybridomas obtained, 76 produced immunoglobulin and eight of them were found to react with the receptor molecule. Only one of the positive clones secreted IgG whereas the other seven produced IgM. The complexes of receptor and antibodies were identified by sucrose density gradient centrifugation. All seven monoclonal antibodies tested reacted with the 90 000 mol. wt. form of the receptor but not with the 40 000 mol. wt. form that contains the steroid and DNA binding domains. None of the monoclonal antibodies interfered with the binding of the receptor to DNA cellulose, thus suggesting that the antigenic determinants are located in a region of the receptor that is not directly implicated in either steroid binding or DNA binding. These antigenic determinants were common to glucocorticoid receptors from several tissues of the rat, whereas glucocorticoid receptors from other species react only with some of the antibodies.     

Monoclonal antibodies against rat T-kininogen: application to radioimmunoassay and immunohistochemistry

Monoclonal antibodies to rat T-kininogen were produced and 9 hybridomas were selected. Radioimmunoassay (RIA) was developed using 125I-labeled T-kininogen and cell walls of Staphylococcus aureus (Zysorbin) for the separation of bound from free ligand, when IgG2a and IgG2b were used. In the case of IgG1 monoclonals, a second antibody (goat anti-mouse IgG) and Zysorbin were used. By this RIA, 1-16 ng T-kininogen/tube showed a linear inhibition curve, and cross reactivities to rat purified LMW- and HMW-kininogens were less than 0.5%, respectively. These monoclonal antibodies were also used for the immunohistochemical staining of the liver to detect T-kininogen in hepatocytes. By using the RIA and immunohistochemical staining, the T-kininogen levels in rat plasma and liver following carrageenin-induced inflammation were estimated. At 3-5 h after the carrageenin injection, when the paw swelling was at its peak, the plasma level of T-kininogen and staining of the liver were slightly increased. T-Kininogen levels in plasma and liver peaked on the 2nd day, when the paw swelling had already decreased. The result indicates that the increase of T-kininogen level in the liver and plasma occurs with a time lag and T-kininogen is not directly involved in the increase of vascular permeability in carrageenin paw edema.     

Isolation and characterization of pepsin-solubilized human basement membrane (type IV) collagen peptides

Native type IV collagen was isolated from human placental tissue by pepsin digestion, fractional salt precipitation, reduction and alkylation, a second pepsin digestion, and chromatography on diethylaminoethyl- and carboxymethyl-cellulose. After denaturation, 10 distinct peptides were isolated from this material by molecular sieve, ion-exchange, and high-performance liquid chromatography. All of the peptides were found to have amino acid compositions characteristic of type IV collagen. Analysis of the eight major peptides by amino-terminal amino acid sequencing and by cyanogen bromide and tryptic peptide mapping has revealed the manner in which they are derived from type IV collagen. Pepsin liberates two large peptides by attacking non-triple-helical regions, one derived from the alpha 1 (IV) chain (F2, Mr 90 000) and one derived from the alpha 2 (IV) chain (F3, Mr 75 000). The alpha 1 (IV)-derived F2 peptide is also represented in the pepsin digest by amino-terminal and carboxy-terminal subfragments [F4c (Mr 41 000) and F4a (Mr 60 000)], as is the alpha 2 (IV)-derived F3 peptide [F5 (Mr 28 000) and F4b (Mr 50 000), respectively]. These findings indicate that the molecular regions from which the larger peptides are derived in themselves contain pepsin-sensitive (non-triple-helical) domains. In addition, several of the peptides examined were found to be present in two slightly different forms, suggesting that closely adjacent pepsin-sensitive sites often exist within the type IV collagen molecules. The methods outlined here provide a reliable means by which identifiable type IV collagen peptides can be isolated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the transmembrane disposition of the neural cell adhesion molecule N-CAM. The use of liposome-inserted radioiodinated N-CAM to study its transbilayer orientation

The transmembrane orientation of the polypeptide chains present in preparations of adult and neonatal mouse N-CAM was studied using, as a model system, liposome-inserted purified N-CAM preparations. N-CAM purified from adult or neonatal mouse brain was 125I-labeled and reconstituted into artificial lipid vesicles. After trypsin digestion, the peptides that remained associated with the liposomes were isolated by floatation of the vesicles on sucrose gradients. In control experiments the liposomes were lysed before trypsin treatment. Large, overlapping peptides were obtained after this treatment, several of which were protected by the liposome membrane. Sialic-acid-bearing peptides were revealed by their sensitivity to neuraminidase. To distinguish between peptides corresponding to intracellular or extracellular domains use was made of the P61 and H28.123 monoclonal antibodies, which recognize determinants located on the cytoplasmic and the extracellular part of the molecules respectively. There was no indication that the N-CAM chains were inserted in an inside-out configuration. Peptides protected from trypsin attack by the liposomes and recognized only by P61 had Mr values of 92 000, 42 000 and 35 000. The H28.123 determinant could be mapped to a 32 000-Mr peptide located close to the membrane at the vesicle's exterior. The bulk of the sialic acid seemed to be carried by a rather short sequence distal to the H28.123-reactive peptide but at some distance from the N terminus. Fragments of very similar Mr were generated from young and adult material. However, a 45 000-Mr peptide from neonatal N-CAM appeared to migrate in the higher-Mr region of sodium dodecyl sulfate/polyacrylamide gels in its fully sialylated form. It is concluded that (a) identical polypeptide chains are present in young and adult preparation, (b) the 180 000-Mr, 140 000-Mr and 120 000-Mr chains differ by the length of their cytoplasmic extensions and (c) the largest cytoplasmic sequences have a Mr close to 90 000. A tentative linear model of the transmembrane topography of the N-CAM polypeptides is presented.     

Polypeptide heterogeneity of hamster and calf fibronectins.

The adhesive glycoprotein fibronectin has been isolated from fresh hamster plasma by affinity chromatography on gelatin coupled to Sepharose beads by the method of Engvall & Ruoslahti [Int. J. Cancer (1979) 20, 1-5]. Polyacrylamide-gel electrophoresis of material heated in sodium dodecyl sulphate and 2-mercaptoethanol shows two prominent polypeptide subunits of approx. mol.wts. 215 000 and 200 000, with variable amounts of lower-molecular-weight fragments. The unexpected polypeptide heterogeneity of different preparations of hamster fibronectins and bovine serum fibronectin is shown to be partly an artefact and is generated during isolation and storage of purified fibronectin. Treatment of each hamster fibronectin subunit or a smaller fragment of approx. mol.wt. 140 000 with thermolysin or trypsin after radioiodination produces similar patterns of tyrpsine-containing peptides, indicating similar primary amino-acid sequences. Antibodies raised against the major subunits of hamster plasma fibronectin were coupled to Sepharose beads and used in conjunction with gelatin affinity chromatography to isolate fibronectins extracted with urea from baby-hamster kidney (BHK) cells and present in the long-term culture medium of these cells. The cell and medium fibronectins are similar to hamster plasma fibronectin in amino-acid and carbohydrate composition and also produce very similar peptide 'maps'. We conclude that the various forms of hamster fibronectins are structurally analogous in agreement with indistinguishable biological properties in mediating the substance adhesion of BKH cells [Pena & Hughes (1978) Cell Biol. Int. Rep. 3, 339-344].     

Demonstration of structural differences between the two subunits of human-plasma fibronectin in the carboxy-terminal heparin-binding domain

Structural differences between the two subunits of human plasma fibronectin were studied by analyzing the carboxy-terminal heparin-binding domain (Hep-2). Two fragments (29 kDa and 38 kDa) derived from the Hep-2 domain were purified from thermolysin-digested human plasma fibronectin. Identical NH2-terminal sequences were obtained for both fragments through 16 Edman cycles. Neither domain contained the 90-amino-acid extra domain which is predicted by cDNA analysis of the cellular form of fibronectin. We have examined the primary structures of the 29-kDa and 38-kDa Hep-2 domains produced from the two chains of plasma fibronectin by analyzing the tryptic peptides by fast atom bombardment/mass spectrometry and comparison with the predicted fragments deduced from the corresponding cDNA-derived peptide sequences. Peptides that were unique to each domain were further characterized by microsequence analysis. The two domains showed identical amino acid sequences through 274 residues, followed by a region of variability. The 29-kDa domain contains 279 amino acids with an estimated relative molecular mass (Mr) of 30,460. This domain is located in the heavy chain of plasma fibronectin and contains three repeats of type III sequences plus a portion of the connecting segment (IIICS) region. The 38-kDa domain contains 359 amino acids and one O-linked glycosyl unit for an estimated Mr of 39,263. This domain is from the light chain of plasma fibronectin and contains four repeats of type III sequences with the deletion of the entire 120-amino-acid IIICS area. Secondary structure analysis by Chou/Fasman and circular dichroism reveals extensive beta-sheet structure for these domains. Key sulfhydryl and glycosylation sites are located near the mRNA splice junctions for the two chains. It is postulated that the splice junctions are adjacent to a flexible domain joining two regions of extensive beta-sheet structure.     

Influence of carbohydrate moieties on the immunogenicity of human immunodeficiency virus type 1 recombinant gp160.

The role of carbohydrates in the immunogenicity of human immunodeficiency virus type 1 (HIV-1) glycoproteins (gp160 and gp120) remains poorly understood. We have analyzed the specificity and neutralizing capacity of antibodies raised against native gp160 or against gp160 deglycosylated by either endo F-N glycanase, neuraminidase, or alpha-mannosidase. Rabbits immunized with these immunogens produced antibodies that recognized recombinant gp160 (rgp160) from HIV-1 in a radioimmunoassay and in an enzyme-linked immunosorbent assay. Antibodies elicited by the different forms of deglycosylated gp160 were analyzed for their reactivity against a panel of synthetic peptides. Compared with anti-native gp160 antisera, serum reactivity to most peptides remained unchanged, or it could increase (peptide P41) or decrease. Only antibodies raised against mannosidase-treated gp160 failed to react with a synthetic peptide (peptide P29) within the V3 loop of gp120. Rabbits immunized with desialylated rgp160 generated antibodies which recognized not only rgp160 from HIV-1 but also rgp140 from HIV-2 at high titers. Although all antisera produced against glycosylated or deglycosylated rgp160 could prevent HIV-1 binding to CD4-positive cells in vitro, only antibodies raised against native or desialylated gp160 neutralized HIV-1 infectivity and inhibited syncytium formation between HIV-1-infected cells and noninfected CD4-positive cells, whereas antibodies raised against alpha-mannosidase-treated gp160 inhibited neither virus replication nor syncytium formation. These findings indicate that the carbohydrate moieties of gp160 can modulate the specificity and the protective efficiency of the antibody response to the molecule.     

Different beta 1-integrin collagen receptors on rat hepatocytes and cardiac fibroblasts

Detergent extracts of primary rat hepatocytes and neonatal cardiac fibroblasts were applied to collagen type I-Sepharose in the presence of 1 mM MnCl2. Elution of bound proteins by 10 mM EDTA yielded one beta 1-integrin heterodimer from hepatocytes with an Mr of 180,000/115,000 under nonreducing conditions. Two beta 1-integrins with Mr's (nonreduced) of 180,000/115,000 and 145,000/115,000 could be isolated from surface-iodinated fibroblasts. A monoclonal antibody, 3A3, directed against the rat homolog of the human integrin VLA-1, precipitated the affinity-purified Mr 180,000/115,000 heterodimer, establishing the relatedness of the Mr 180,000 subunit to the alpha 1-chain of the beta 1-integrin subfamily. Both the alpha 1 beta 1-integrin and the 145,000/beta 1-integrin heterodimers bound specifically to Sepharose beads derivatized with the collagen fragment alpha 1(I) CB3, which lacks RGD sequences. Immunofluorescence staining using the 3A3 monoclonal antibody revealed that the rat alpha 1 beta 1-integrin was present at focal adhesion sites of fibroblasts grown on native collagen type I- but not on fibronectin-coated substrates, although both types of substrates supported the formation of beta 1-integrin containing focal adhesions. Similarly, hepatocytes cultured on substrata coated with collagen type I (but not fibronectin) were stained in a patchy pattern localized to the cell periphery by 3A3 IgG. Furthermore, 3A3 IgG completely inhibited the attachment of hepatocytes to collagen type I, whereas under identical conditions the attachment of fibroblasts to these substrates was inhibited only by approximately 40%. The attachment of both hepatocytes and cardiac fibroblasts to fibronectin was unaffected by the presence of the 3A3 antibody. Collectively these data show that a rat homolog of the human VLA-1 heterodimer both biochemically and functionally fulfills the criteria of a single collagen receptor on rat hepatocytes. In contrast, rat cardiac fibroblasts utilize two different collagen-binding integrins to adhere to collagen, one of which is the rat homolog of the human VLA-1 heterodimer. Furthermore alpha 1(I) CB3 contains cell binding sites for beta 1-integrins.     

Monoclonal antibodies reacting with isotypic and/or allotypic determinants of human IgG

Mice were immunized with purified human IgG myeloma proteins and hybridomas were prepared using their spleen cells. 1817 of the hybridomas secreted anti-Ig antibodies. Several of them detected subclass-associated determinants. Eight different specificities could be distinguished. The number of hybridomas in each category were the following: 3 anti-IgG1 (1.8% of all anti-Ig clones) 5 anti-IgG2 (0.3% of all anti-Ig clones) 2 anti-IgG3 (2.5% of all anti-Ig clones) 3 anti-IgG4 (12% of all anti-Ig clones) 12 anti-IgG1, IgG2, IgG3 70 anti-IgG1, IgG2, IgG4 2 anti-IgG2, IgG4 7 anti-IgG2, IgG4 (one out of five myelomas).     

Domain structure of rat liver carbamoyl phosphate synthetase I

Independently folded structural domains of rat liver carbamoyl phosphate synthetase I have been identified by partial proteolytic cleavage under nondenaturing conditions. The pattern of fragments produced was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal sequences of the fragments were determined by automated Edman degradation. Comparison of these fragment sequences with the sequence of the intact protein allowed alignment of the fragments. The hydrolysis of carbamoyl phosphate synthetase I (Mr 160,000) by either trypsin or elastase proceeded in two stages, with two alternative routes of degradation for elastase. The alignment of the final tryptic fragments from the NH2 terminus to the COOH terminus was: Mr 87,000 fragment-Mr 62,000 fragment-group of small peptides. The alignment of the final elastase fragments was: Mr 37,000 fragment-Mr 108,000 fragment-group of small peptides. The rates of cleavage were affected by the presence of the substrate ATP or the positive allosteric effector N-acetylglutamate; the preferred route of elastase cleavage was also affected. In addition to providing a map of the carbamoyl phosphate synthetase I domains and preliminary information on the interaction of substrates with these domains, the present studies provide further support for the proposal that domains serve as units of protein evolution since the 37-kDa fragment encompasses the region of the rat liver synthetase that is homologous to the 40-kDa subunit of the Escherichia coli synthetase.