Bovine Conglutinin Research Articles

Analysis of multiple antigenic determinants of the bovine conglutinin molecule using different synthetic peptides.

Twenty-three overlapping peptides corresponding to the complete amino acid sequence of the bovine conglutinin molecule were synthesized on the basis of the sequence data to analyze antigenic determinants. Of the synthetic peptides tested, peptides 31-50, 166-185, and 241-260 were highly reactive with rabbit antibodies against native conglutinin, whereas peptides 1-20, 16-35, 31-50, 76-95, 136-155, 151-170, 181-200, 271-290, 301-320, and 316-335 were less reactive with the antibodies. Peptides 16-35, 31-50, 46-65, 76-95, 136-155, 151-170, 181-200, 271-290, 301-320, and 316-335 were less reactive with the antibodies. Peptides 16-35, 31-50, 46-65, 76-95, 91-110, 106-125, 151-170, 166-185, 211-230, 226-245, and 286-305 inhibited the binding of native conglutinin to mannan. On the other hand, all of these synthetic peptides elicited rabbit antibodies reactive with native conglutinin. Antibodies to peptide 241-260 showed the highest reactivity with native conglutinin. Of these anti-peptide antibodies, however, only antibodies to synthetic peptide 46-65 inhibited effectively the binding of conglutinin to mannan. Antibodies to the remaining peptides failed to block the binding at the highest concentrations tested. These results suggest that the multiple antigenic determinants associated with both ligand binding and immunogenicity may be located on the conglutinin molecule and that the predominant antigenic determinant responsible for binding saccharides may be located in the vicinity of the region 46-65.

Differentiation of Conglutination Activity and Sugar-Binding Activity of Conglutinin after Removal of NH 2-Terminal 54 Amino Acid Residues by Endogenous Serine Protease(s)

Bovine conglutinin, which is a serum lectin specific for N-acetylglucosamine, was purified from heat-inactivated bovine serum by Sepharose 4B-mannan affinity chromatography. Without prior heat inactivation, however, the same affinity chromatography yielded a modified conglutinin, which retained sugar-binding, but not conglutination activity. The modified lectin was designated conglutinin-N. The conglutinin-N subunit (38 kDa) was smaller than that of conglutinin (45 kDa). The apparent molecular mass was also reduced from 1000 (conglutinin) to 800 kDa (conglutinin-N). Conglutinin-N and conglutinin reacted equally with a rabbit anti-conglutinin antiserum. Comparison of the NH 2-terminal sequences of conglutinin-N with the primary structure of conglutinin [Lee, Y-M., Leiby, K. R., Allar, J., Paris, K., Lerch, B., and Okarma, T. B. (1991) J. Biol. Chem. 266, 2715-2723] revealed that the removal of the 54 NH 2-terminal amino acids of conglutinin produced conglutinin-N. This conversion of conglutinin to conglutinin-N was catalyzed by the eluate fraction of the first affinity chromatography from untreated bovine serum and was inhibited by serine protease inhibitors such as [4-aminophenylimethanesulfonyl fluoride and C1 inhibitor. Conglutinin-N had neither conglutination activity nor binding activity to the sensitized erythrocyte-solid phase iC3b complex (EAiC3b), although it retained the binding activity to mannan in solution and the original binding specificity for N-acetylglucosamine. Cross-linking showed that conglutinin-N formed trimers of the 38-kDa subunits, whereas conglutinin formed higher multimers of 9-18 mer. These results indicated that deleting the NH 2-terminal portion containing three cysteine residues by endogenous serine protease(s) produced some conformational changes in the entire molecule, which resulted in the loss of binding activity to EAiC3b and retention of the sugar-binding property.

Cloning and sequencing of a cDNA coding for bovine conglutinin

Cloning and sequencing of a cDNA coding for bovine conglutinin

An Immune Complex Selective Affinity Column Utilizing Site-Specific Attachment of Bovine Conglutinin

A simple method to isolate immune complexes by column chromatography is described. The immune complex affinity column was constructed by the site-specific attachment of bovine conglutinin to agarose. Covalent attachment of conglutinin to agarose was achieved via hydrazone chemistry, which reacts aidehydes of oxidized oligosaccharides in the collagenous domain of conglutinin with hydrazide functional groups in the solid support. The constructed conglutinin affinity column captured complement-fixed model immune complexes of heat-aggregated human IgG and more classical complexes of chicken ovalbumin-antichicken ovalbumin. Neither the nonfixed immune complexes nor their individual components were retained by the column. The captured material was specifically eluted with EDTA without the use of low pH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and protein sequence analysis of the eluates revealed the presence of the expected individual components, verifying that both antibody and antigen used to prepare the soluble immune complexes wore recovered from the conglutinin column. The advantages of this approach over traditional methods of immune complex isolation and characterization are discussed.

S12.5 Cloning and sequencing of a cDNA coding for bovine conglutinin

S12.5 Cloning and sequencing of a cDNA coding for bovine conglutinin

Structural similarity between lung surfactant protein D and conglutinin

Preparations of bovine lung surfactant D (SP-D) and conglutinin were examined by electron microscopy, gel-filtration and SDS/PAGE. SP-D is composed of non-covalently linked subunits, of 160 kDa, which each contain three, disulphide-linked, 44-kDa polypeptide chains. In the electron microscope a single 160-kDa subunit of SP-D appears as a 45.8 +/- 3-nm-long rod connected to a small globular 'head'. Particles were also seen which correspond to non-covalently linked dimers, trimers and tetramers of the 160-kDa monomer subunit of SP-D. The tetramer structure contains 12 polypeptide chains and is very similar to the electron microscopy images and model reported by Strang et al. [Strang, C. J., Slayter, US., Lachmann, P. J. and Davis, A. E. (1986) Biochem. J. 236, 3811-389] for bovine conglutinin in which four 160-kDa subunits are disulphide-linked to give a molecule of expected molecular mass of 528 kDa. This study confirmed the findings by Strang et al. in the above paper for intact conglutinin and also emphasised that the rod-like structures, of length 37.6 +/- 3.7 nm, seen in the conglutinin subunits were significantly shorter than those in SP-D despite the close similarity in amino acid sequence (79% identify) and chain length between the two proteins. In addition, a truncated form of conglutinin was found in the conglutinin preparations, due to limited proteolysis of the Arg-Ala bond at position 54 in the 44-kDa chains. These truncated conglutinin chains yield a subunit composed of three shortened, non-disulphide-linked, chains and this subunit appears as a monomer with a rod length of 34.2 +/- 2.8 nm in the electron microscope. On gel-filtration, a proportion of the SP-D preparation behaved, as expected, as a molecule with an apparent molecular mass of 600 kDa. The remainder of the SP-D preparation behaved as aggregated material with a molecular mass greater than 900 kDa which yielded no distinct structures in the electron microscope. Intact conglutinin was eluted at a position greater than 900 kDa but yet provided clear electron microscopy images of the tetramer structure described above.(ABSTRACT TRUNCATED AT 400 WORDS)

Localization of the receptor-binding site in the collectin family of proteins.

Collectin receptor (Clq receptor) has been shown to bind human Clq, mannose-binding protein (MBP), lung surfactant protein A (SP-A) and bovine conglutinin. These ligands have a similar ultrastructure, each consisting of collagenous and globular domains, but do not show a high degree of sequence similarity. For Clq and SP-A, it has been shown that both bind to cell-surface-expressed receptor(s) via their collagenous regions and this is likely to be the case with the other ligands. Within the collagenous region, near the 'bend' region of the collagen triple helix in Clq, MBP and SP-A, a cluster of similar charged residues is observed. This region has been suggested to be associated with receptor binding. A similar region of charge density occurs close to the N-terminus of conglutinin. In this paper we describe a truncated form of conglutinin, which has 55 amino acids missing from the N-terminus and does not bind to the collectin receptor. The results presented here strongly indicate that receptor-ligand interaction is mediated via the N-terminal region of conglutinin, consistent with the earlier proposal for the binding site.

The cDNA cloning of conglutinin and identification of liver as a primary site of synthesis of conglutinin in members of the Bovidae

Bovine conglutinin is a collagen-like, C-type, plasma lectin which belongs to the group of proteins called 'collectins'. Two inosine-containing oligonucleotides were synthesized, based on the published protein sequence for bovine conglutinin [Lee, Leiby, Allar, Paris, Lerch and Okarma (1991) J. Biol. Chem. 266, 2715-2723], and PCR on target DNA from a bovine liver lambda gt 11 cDNA library yielded a product of the expected size of 210 bp. Screening of the library with this cDNA fragment identified a single positive clone, with an insert of 0.9 kb, coding for bovine conglutinin from residue 70 to the C-terminus. The 5' cDNA sequence, encompassing 150 bp of the 5' non-translated sequence plus the sequence encoding the leader peptide and the N-terminal residues 1-70, was completed by the use of PCR techniques. The cDNA sequence of bovine conglutinin showed 86% identity with that of bovine lung surfactant protein D (SP-D), and the derived amino acid sequence of bovine conglutinin showed 78% identity with that of bovine SP-D, which included complete identity of the leader-peptide sequences. The amino acid sequence derived from the cDNA sequence differs from the published protein sequence at four positions. Northern-blot analysis on total RNA, purified from various tissues from cattle, sheep, humans, rats and mice, showed that a strong signal of approx. 1.8 kb is present in bovine liver RNA. A weak signal of similar size was also observed in sheep liver, but not in human, rat and mouse livers. A weak signal, also of 1.8 kb, is present in the lung RNAs of all the species tested. The signals from the lung tissues are likely to be due to the cross-hybridization of the bovine conglutinin cDNA to the SP-D mRNAs of the respective species. The finding of significant signals in only the bovine and sheep liver RNA samples is indicative that serum conglutinin may be present in significant amounts only in members of the Bovidae (the family encompassing cattle, antelopes, sheep and goats) and closely related species.

Purification and characterization of a bovine serum lectin (CL-43) with structural homology to conglutinin and SP-D and carbohydrate specificity similar to mannan-binding protein

A previously undescribed bovine serum lectin (designated CL-43) was identified by its Ca(2+)-dependent binding to mannan and by its molecular mass of 43 kDa under reducing conditions on SDS-PAGE. The lectin was isolated by polyethylene glycol precipitation, affinity chromatography on mannan-Sepharose (followed by elution with EDTA), and absorption on Sepharose-4B-coupled rabbit anti-bovine Ig (to remove anti-mannan antibodies). Fractions containing the lectin were reapplied to mannan-Sepharose. Bound conglutinin was eluted with GlcNAc, and then the 43-kDa lectin, together with mannan-binding protein (MBP), was eluted with mannose. The 43-kDa lectin was separated from MBP by ion exchange chromatography on Mono-Q. On SDS-PAGE under nonreducing conditions the lectin showed a molecular mass of 120 kDa. On gel chromatography under nondissociating conditions the protein was eluted at a volume corresponding to a molecular mass of approximately 750 kDa. Amino acid analysis showed the presence of hydroxyproline and hydroxylysine and a high content of glycine (24.3%) indicating the presence of a collagen-like structure. This was supported by the susceptibility of the protein to collagenase digestion. The designation CL-43 was chosen since this molecule appears to belong to the collectins, i.e. proteins with collagen structure and lectin activity. The N-terminal sequence (27 amino acids) showed 56% identity with bovine SP-D and 44% identity to bovine conglutinin. An inhibition assay with biotinylated CL-43, using solid-phase mannan as ligand, revealed the following carbohydrate inhibition pattern: mannose and ManNAc > fucose > GlcNAc > glucose and maltose > galactose > lactose >> GalNAc. We conclude that CL-43 is a circulating lectin, with structural similarities to bovine conglutinin and SP-D, and a ligand binding profile resembling that of MBP.

Cloning and Sequencing of a cDNA Coding for Bovine Conglutinin

A 912 bp bovine cDNA fragment encoding bovineconglutinin was amplified by the RT-PCR technique. cDNA clones encoding the bovine conglutinin were isolated from a bovine liver cDNA library using a specific probe obtained from the PCR product. These cDNAs carry an insert of 1113 bp coding for a protein of 371 amino acid residues with a signal peptide of 20 residues. The deduced amino acid sequence of cDNA agrees with that determined by conventional amino acid sequence analysis. Two polyadenylation signal sequences were detected in the DNA sequence downstream of the 3′ end of the gene. Southern blot analysis of total bovine genomic DNA indicated that there is only one copy of the gene encoding bovine conglutinin. Northern blot analysis of bovine tissues showed that conglutinin mRNA of about 1.5 kb is expressed in the liver and also slightly in the lung.

1] Complement factor I and cofactors in control of complement system convertase enzymes

This chapter describes the complement factor I and cofactors in control of complement system convertase enzymes. Complement factor I was first described and partially characterized in 1966–1968 as an enzyme involved in the physiological degradation of the major complement protein C3. It was previously named conglutinogen-activating factor (KAF), because its action on C3 exposed a binding site on a C3 fragment for the bovine protein conglutinin. Other former names include C3b inactivator, C3b INA, and C3b/C4b inactivator. The physiological substrates of factor I are produced only when the complement system is activated. Activation of the classical pathway of the complement system is generally mediated by binding of the C1 complex to a target, such as immune complexes or micro-organisms. The role of factor I is to control the activities of the convertase enzymes, C4b2a and C4b2a3b, by proteolytic cleavage of the C4b and C3b subunits of these enzymes. Before C3b or C4b can be cleaved by factor I, they must bind to one of several complement control proteins to form a non-covalent complex.

Biochemical and ultrastructural studies of the C-type lectin bovine conglutinin

The aim of this study was to correlate the supramolecular organization of conglutinin (BK) with its primary and tertiary structure and to gain more knowledge of functionally important regions of the molecule. BK analyzed by SDS-PAGE under standard reducing conditions (40 mM DTT) showed a major band at 43 kDa and weaker bands at 86 and 180 kDa. In contrast, reduction with 6–50 mMl-cysteine resulted in 37-kDa subunits indicating the presence of intrachain disulfide bonds within this subunit. Hydroxylamine treatment indicated presence of ester bonds in the 86- and 180-kDa subunits. Collagenase digestion and SDS-PAGE under reducing and nonreducing conditions resulted in bands of 20 and 15 kDa, respectively, indicating the presence of intrachain, rather than interchain, disulfide bonds in the carboxy terminus. Deglycosylation and glycan differentiation analysis of BK revealed the presence of O-linked glycans of GalNAc and α(2–3) linked sialic acid type, whereas no N-linked glycans were demonstrated. Binding experiments with GlcNAc-gold suggested that multivalency is required for carbohydrate binding to BK. Electron microscopy showed mostly tetramers, 96 nm in diameter, but also mono-, di-, and trimers were seen. The tetramers consisted of 40-nm strands, each with a peripheral globular head composed of subunits and connected to a common central lobe built from four ring-formed structures. The strands occasionally showed two bends, one close to the central lobe and another 25 nm from the lobe. These bends most likely correspond to the interrupted Gly-Xaa-Yaa repeats at residues 38 and 123.

Purification, subunit characterization and ultrastructure of three soluble bovine lectins: conglutinin, mannose-binding protein and the pentraxin serum amyloid P-component.

Conglutinin and mannose-binding protein (MBP) are members of the C-type lectins which are widely present in mammalian plasma. Serum amyloid P-component (SAP) is a member of the pentraxin family with lectin properties. A scheme for the partial purification of all three lectins by carbohydrate affinity chromatography and selective elution was developed. The purification was monitored by SDS-PAGE, Western blotting and electron microscopy. Binding of the lectins to Sephadex-iC3b, their collagenase sensitivity, and the size and antibody reactivity of their subunits was investigated. The demonstration, by SDS-PAGE, of 25-kDa subunits, which were unaffected by collagenase treatment but bound to Sephadex-iC3b and antibodies to human SAP, indicated the existence of bovine SAP. Bovine conglutinin (BK) also showed calcium-dependent binding to Sephadex-iC3b, whereas bovine MBP did not. The binding of BK was inhibitable with GlcNAc. A 3000-fold increase in BK activity (ELISA) was obtained in eluates from Sephadex-iC3b. SDS-PAGE analyses of BK and MBP revealed subunits with an Mr of 43 kDa and 30 kDa, respectively. These subunits were sensitive to collagenase treatment which reduced the Mr to 20 kDa. Electron micrographs revealed a prominent flexible tetramer molecule (diameter 96 nm) in the BK preparations, a predominantly hexameric structure (diameter 30 nm) in the MBP preparations, and single annular pentameric disc-like molecules (diameter 11 nm) in the SAP preparations.

Inhibition of human immunodeficiency virus-1 infection by human conglutinin-like protein: in vitro studies.

The lectin‐like protein analogous to bovine conglutinin was purified from human serum. The carbohydrate‐binding ability of conglutinin‐like protein was inhibited by D‐mannose, N‐acetylglucosamine and L‐fucose as well as by mannan‐containing oligosaccharides. By applying a lectin‐based ELISA system it was demonstrated that conglutinin‐like protein binds to human immunodeficiency virus‐1 (HIV‐1) glycoprotein 120 (gpl20) via its carbohydrate binding site. In vitro experiments with T‐lymphoblastoid CEM cells revealed that conglutinin‐like protein abolishes infection by HIV‐1; a 50% cytoprotective concentration of 23.9 μg/ml was measured. These findings demonstrate that human conglutinin‐like protein binds to HIV‐gp120 and inhibits, under the described in vitro conditions, CEM cell infection.

Calcium-dependent and calcium-independent signals in the conglutinin-binding assay (KgBa) for immune complexes: Influence of anti-collagen-antibodies

A solid phase ELISA conglutinin-binding assay (KgBa) was evaluated for the detection of circulating immune complexes. ELISA wells were coated with purified bovine conglutinin and incubated with test sera. Bound IgG was detected with enzyme labelled anti-immunoglobulin. Heat aggregated IgG which had been “solubilized” (i.e., complement treated by incubation with serum) was employed as a reference. The binding of the complement-reacted IgG to solid phase conglutinin was found to be calcium-dependent and inhibitable with N- acetyl- D- glucosamine (GlcNAc). Prolonged incubation (4 days) of aggregated IgG with serum at 37°C abolished the binding to conglutinin, a finding consistent with the complete degradation of deposited C3b to C3c and C3d. The solubilized IgG that bound to solid phase conglutinin was found by gel chromatography to be of high molecular weight (>600 kDa). Binding of IgG to solid phase bovine conglutinin was also observed to a variable degree in normal and pathological sera. However, in this situation the IgG binding was largely calcium-independent, was not inhibited by GlcNAc and did not decrease after prolonged incubation of the serum at 37°C. The reactive IgG eluted on gel chromatography at the position of monomeric IgG suggesting binding via the antigen binding sites. Binding of this IgG was inhibited by both collagen type II and purified conglutinin. These observations suggest that the assay detects cross-reacting autoantibodies against collagen epitopes, or, alternatively, antibodies against the dietary antigen, bovine conglutinin.

Primary structure of rat pulmonary surfactant protein D. cDNA and deduced amino acid sequence.

Surfactant protein D (SP-D) is a carbohydrate-binding glycoprotein containing a collagen-like domain that is synthesized by alveolar type II epithelial cells. The complete primary structure of rat SP-D has been determined by sequencing of a cloned cDNA. The protein consists of three regions: an NH2-terminal segment of 25 amino acids, a collagen-like domain consisting of 59 Gly-X-Y repeats, and a COOH-terminal carbohydrate recognition domain of 153 amino acids. There are 6 cysteine residues present in rat SP-D: 2 in the NH2-terminal noncollagenous segment and 4 in the COOH-terminal carbohydrate-binding domain. The collagenous domain contains one possible N-glycosylation site. The protein is preceded by a cleaved, NH2-terminal signal peptide. SP-D shares considerable homology with the C-type mammalian lectins. Hybridization analysis demonstrates that rat SP-D is encoded by a 1.3-kilobase mRNA which is abundant in lung and highly enriched in alveolar type II cells. Extensive homology exists between rat SP-D and bovine conglutinin.

Human conglutinin-like protein inhibits infection by the human immunodeficiency virus-1 in vitro

In summary the lectin-like protein analogous to bovine conglutinin was purified from human serum. Using a lectin-based ELISA system, it was demonstrated that conglutinin-like protein binds to human immunodeficiency virus-1 (HIV1) glycoprotein 120 (gp 120) via its carbohydrate binding site. In vitro experiments with T-lymphoblastoid CEM cells revealed that conglutinin-like protein abolishes infection by HIV1; a 50 % cytoprotective concentration of 23.9 μg/ml was measured.

Primary structure of bovine conglutinin, a member of the C-type animal lectin family.

We report the complete amino acid sequence of bovine conglutinin obtained by structural characterization of peptides derived from the protein by various chemical and enzymatic fragmentation methods. The protein consists of 351 amino acid residues including 55 apparent Gly-X-Y repeats with two interruptions. This 171-residue-long collagenous domain separates a short noncollagenous NH2-terminal region of 25 residues from the 155-residue-long globular COOH terminus revealing the structural relation of conglutinin with mannose-binding proteins, pulmonary surfactant-associated proteins, and a complement component C1q. Eight hydroxylysine residues were found in the collagenous domain. All of these hydroxylysine residues which occupy a Y position in a Gly-X-Y triplet are possible glycosylation sites since no phenylthiohydantoin amino acid was identified in automated Edman degradation cycles corresponding to these sites. The noncollagenous COOH domain of conglutinin, on the other hand, contains a carbohydrate recognition domain which shares substantial sequence homology with C-type animal lectins. Conglutinin has the greatest sequence similarity with mannose-binding proteins and pulmonary surfactant-associated proteins.

Conglutinin Binds The HIV‐1 Envelope Glycoprotein gpl60 and Inhibits its Interaction with Cell Membrane CD4

The highly glycosylated envelope glycoprotein (gp 160) of human immunodeficiency virus (HIV) interacts with the CD4 molecule present on the membrane of CD4+ cells and is involved in the pathobiology of HIV infection. Lectins bind glycoproteins through non-covalent interactions with specific hexose residues. The mammalian C-type lectin bovine conglutinin was examined for its ability to interact with recombinant gp160 (rgp160) produced in vaccinia virus-infected BHK21 cells. Specific binding of conglutinin to rgp160 was demonstrated by ELISA. The interaction of bovine conglutinin with rgp160 was calcium-dependent, which is characteristic of the binding of a C-type lectin to its ligand, and the binding was inhibited in a dose-dependent manner with N-acetyl-D-glucosamine. Deglycosylation of rgp160 abrogated the conglutinin binding. In addition, conglutinin exerted a dose-dependent inhibition of the binding of rgp160 to the CD4 receptor on CEM 13 cells, as demonstrated by FACS analyses. These results indicate that conglutinin may inhibit the infection with HIV-1 through its interaction with the viral envelope glycoprotein.

A library of Oligosaccharide Probes (Neoglycolipids) from N-Glycosylated Proteins Reveals That Conglutinin Binds to Certain Complex-type as Well as High Mannose-type Oligosaccharide Chains

This report describes the preparation of a library of oligosaccharide probes (neoglycolipids) from N-glycosylated proteins, characterization of the probes by liquid secondary ion mass spectrometry, and investigation of their reactions with 125I-labeled bovine serum conglutinin by chromatogram binding assays. The results, together with additional binding studies using neoglycolipids derived from purified complex type bi-, tri-, and tetraantennary oligosaccharides from urine, or their glycosidase-treated products, have shown that the combining specificity of conglutinin includes structures not only on high mannose-type oligosaccharides but also on hybrid- and complex-type chains. With high mannose-type oligosaccharides there is increased reactivity from the Man5 to the Man8 structures, indicating a preference for the terminal Man alpha 1-2 sequence. With complex- and hybrid-type oligosaccharides, the requirements for binding are the presence of nonreducing terminal N-acetylglucosamine or mannose residues, but the presence of a bisecting N-acetylglucosamine residue may inhibit binding. From these results it is deduced that the reactivity of conglutinin with the complement glycopeptide iC3b rather than the intact glycoprotein C3 is due to the oligosaccharide accessibility rendered by proteolysis in the complement cascade.