Bovine serum conglutinin is a lectin which binds non-reducing terminal N-acetylglucosamine, mannose and fucose residues.

Carbohydrate recognition by bovine serum conglutinin has been investigated by inhibition and direct binding assays using glycoproteins and polysaccharides from Saccharomyces cerevisiae (baker's yeast), and neoglycolipids derived from N-acetylglucosamine oligomers, mannobiose and human milk oligosaccharides. The results clearly show that conglutinin is a lectin which binds terminal N-acetylglucosamine, mannose and fucose residues as found in chitobiose (GlcNAc beta 1-4GlcNAc), mannobiose (Man alpha 1-3Man) and lacto-N-fucopentaose II [Fuc alpha 1-4(Gal beta 1-3)GlcNAc beta 1-3Gal beta 1-4Glc] respectively.     

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.     

Isolation and characterization of mannan-binding proteins from chicken liver

Two binding proteins which recognize and bind mannose and N-acetylglucosamine (mannan-binding proteins, MBP) have been isolated from chicken liver to near homogeneity mainly by affinity chromatography on a column of Sepharose 4B-mannan. The neutral binding protein (pI 7.0), which has a high glycine content, is an analog of mammalian liver MBP (F-I). F-I consists of a series of proteins composed of two subunits of 28,000 (A) and 32,000 (B) Da. The proteins have molecular weights ranging from 280,000 to 740,000 and subunit compositions ranging from 6A + 4B to 5A + 19B. With increasing molecular weight the specific activity of mannan binding increases gradually, accompanied by a slight change in specificity to a preference for mannose rather than N-acetylglucosamine. The acidic binding protein (pI 5.1) is a glycoprotein with a high glutamic acid content (F-II). The molecular weight of F-II was estimated to be 640,000, and it is composed of single subunits of 41,000 Da. The two MBPs isolated in this study are distinct from the liver lectin specific for N-acetylglucosamine-terminated glycoproteins isolated from the same source [T. Kawasaki and G. Ashwell (1977) J. Biol. Chem. 252, 6536-6543] in chemical properties and binding specificities.     

Carbohydrate-binding proteins of human serum: isolation of two mannose/fucose-specific lectins

Two lectins with specificities for mannose and fucose have been isolated from human serum by affinity chromatography. One mannose-binding protein (MBP 1) has a native Mr of 700,000 with subunits of Mr 32,000 and has specificities for N-acetylglucosamine, N-acetylmannosamine and glucose as well as for mannose and fucose. The other mannose-binding protein (MBP 2) has a native Mr of 200,000 with subunits of Mr 28,000 and is specific only for mannose and fucose. MBP 2 appears to recognize the core sugars of asparagine-linked oligosaccharides as well as the terminal sugars. Both lectins are calcium-dependent, requiring approx. 0.095 mM calcium for half-maximal binding. MBP 1 binds maximally between pH 7-9, whereas MBP 2 has a pH optimum of 6-7. The binding activity of both proteins decreases rapidly below pH 5. The apparent association constants (Ka) for binding to mannon are 2.1 X 10(8) M-1 for MBP 1 and 1.3 X 10(8) M-1 for MBP 2. These data provide further evidence of the complex nature of mammalian carbohydrate recognition systems.     

Localization of the conglutinin binding site on the third component of human complement

The binding site on the human third complement component for bovine conglutinin has been located. C3 fragments were purified to homogeneity by preparative SDS-polyacrylamide-gel electrophoresis. Only the N-terminal 27,000 dalton (Da) fragment of the alpha'-chain and the beta-chain were found to be glycosylated, and the carbohydrate was susceptible to endo-beta-N-acetylglucosaminidase H. This finding indicates that only high mannose or hybrid-type oligosaccharide chains are present on the C3 molecule. Binding to conglutinin was determined by an enzyme-linked immunosorbent assay and occurred with C3b, iC3b, C3c, the alpha-chain, and the 27,000 Da fragment of the alpha'-chain, but not with C3d or the C-terminal 40,000 Da fragment of the alpha'-chain. The beta-chain displayed very weak interaction. Binding to conglutinin could be inhibited by EDTA, N-acetylglucosamine, and to a lesser degree by mannose. Enzymatic removal of the carbohydrate from the C3 molecule abolished binding to conglutinin. It is concluded that bovine conglutinin binds to the carbohydrate moiety located on the N-terminal 27,000 Da polypeptide of the alpha-chain.     

Characterization of the oligosaccharide units of the bovine erythrocyte membrane glycoprotein.

The major glycoprotein of the bovine erythrocyte membrane was purified by extraction of the ghosts with lithium 3,5-diiodosalicylate followed by phenol-water extraction and acidification. The glycoprotein contains 20% protein and 80% carbohydrate by weight and gives a single band on sodium dodecyl sulfate-polyacrylamide gels with an estimated molecular weight of 230000 daltons. The carbohydrate composition of the glycoprotein was determined to be (in residues relative to sialic acid): sialic acid, 1.0; fucose, less than 0.01; mannose, 0.1; galactose, 3.3; N-acetylgalactosamine, 0.9; and N-acetylglucosamine, 2.4. Pronase digestion of the isolated glycoprotein followed by Sephadex G-75 gel filtration resulted in the separation of a small pool of glycopeptides (pool III), which included all of the mannose-containing glycopeptides, from the bulk of the glycopeptide material which was in the void fractions of the column (pool I). Alkaline borohydride treatment released over 95% of the oligosaccharide units in pool I and approximately 30% of the oligosaccharide units in pool III. These oligosaccharides were isolated by gel filtration and ion-exchange chromatography. The oligosaccharides released from pool I had molecular weights of 1100-1400 daltons and contained sialic acid, galactose, and N-acetylglucosamine in molar ratios of 0.5-1:3:2 as well as a partial residue of N-acetylgalactosaminitol. The oligosaccharides released from pool III by alkali had molecular weights of 1300-1600 daltons and contained sialic acid, galactose, N-acetylglucosamine, N-acetylgalactosamine and N-ACETYLgalactosaminitol in molar ratios of 1-2:2:1:1:1. These data indicate that the majority of the oligosaccharide units of the bovine erythrocyte glycoprotein are linked O-glycosidically to the peptide backbone of the molecule.     

Serum lectin with known structure activates complement through the classical pathway

Serum mannan-binding protein (MBP), a lectin specific for mannose and N-acetylglucosamine, was revealed to activate the complement system as measured by passive hemolysis using sheep erythrocytes coated with yeast mannan. In contrast, rat liver MBP, which shares many properties in common with serum MBP, could not activate complement at all. The activation by serum MBP was inhibited effectively by the presence of haptenic sugars and dependent absolutely upon the presence of C4, indicating that the activation is initiated by the sugar binding activity of MBP and proceeds through the classical pathway. The 25 NH2-terminal amino acid sequence of rat serum MBP determined in this study was completely matched with that of MBP-A deduced from cDNA sequence by Drickamer et al. (Drickamer, K., Dordal, M. S., and Reynolds, L. (1986) J. Biol. Chem. 261, 6878-6887), revealing that MBP-A is in fact identical with serum MBP. On the basis of the knowledge of primary structures and physicochemical properties of rat serum and liver MBPs, a possible mechanism of the complement activation by serum MBP is discussed with reference to close similarity in the gross structures of serum MBP and C1q.     

Isolation and characterization of two distinct mannan-binding proteins from rat serum

Two binding proteins, which are specific for mannose and N-acetylglucosamine, were isolated from rat serum to homogeneity. The minor component [serum mannan-binding protein I (S-MBP-I)] was indistinguishable from rat liver mannan-binding protein (L-MBP). S-MBP-I had a molecular mass of about 200 kDa and consisted of about six identical 32-kDa subunits; the molecule had a collagen-like structure, and its properties were identical to those of L-MBP. S-MBP-I was also indistinguishable from L-MBP in immunochemical reactivity. Furthermore, the sequence of 15 NH2-terminal amino acids of S-MBP-I was identical to that of L-MBP, the complete primary structure of which has been elucidated [Drickamer, K., Dordal, M. S., and Reynolds, L. (1986) J. Biol. Chem. 261, 6878-6887; Oka, S., Itoh, N., Kawasaki, T., and Yamashina, I. (1987) J. Biochem. 101, 135-144]. The major component (S-MBP-II) had a molecular mass of about 650 kDa and consisted of about 20 identical 31-kDa subunits; it was immunochemically distinct from L-MBP and S-MBP-I, although the molecule had a collagen-like structure similar to L-MBP and S-MBP-I. Metabolic studies using [3H]leucine showed that S-MBP-II is a typical plasma protein turning over with a half-life of 1.6 days. S-MBP-I was unusual in its late appearance and rapid turnover rate in plasma. These results, together with the fact that L-MBP decayed with biphasic curves, suggest that a part of L-MBP is leaked from liver into plasma in the form of S-MBP-I.     

Cloning and characterization of a cDNA encoding bovine mannan-binding protein

To identify the bovine mannan-binding protein (MBP), a search for the cDNA homologue of human MBP was carried out. cDNA clones encoding bovine MBP were isolated from a bovine liver cDNA library using a cDNA fragment encoding a short collagen region, neck domain and carbohydrate recognition domain of human MBP. The cDNA carried an insert of 747 bp encoding a protein of 249 amino acid (aa) residues with a signal peptide of 19 aa. The mannan-binding protein fraction of bovine serum that eluted with 100 mM mannose from a mannan-Sepharose column was analyzed under reducing conditions by SDS-PAGE. The major band of 33 kDa obtained reacted with anti-human MBP rabbit serum. The partial aa sequence of the purified 33-kDa protein was identical to the aa sequence deduced from the obtained cDNA. Results of the passive hemolysis experiment using sheep erythrocytes coated with yeast mannan suggest that this MBP has the ability to activate complement. Northern blot analysis showed a 1.8-kb mRNA that was expressed only in the liver. Based on results of genomic analysis, this bovine MBP is likely to be a homologue of human MBP and to also have homology to rat and mouse MBP-C which are localized in liver cells rather than to rat and mouse MBP-A found in serum. Alignments of bovine collectins show that bovine MBP cannot be included among the other bovine collectins, such as bovine SP-D, conglutinin and CL-43. Finally, these genomic and biological analyses indicate that the cDNA obtained here encoded a bovine serum MBP.     

Differential recognition of core and terminal portions of oligosaccharide ligands by carbohydrate-recognition domains of two mannose-binding proteins

Two different mannose-binding proteins (MBP-A and MBP-C), which show 56% sequence identity, are present in rat serum and liver. It has previously been shown that MBP-A binds to a range of monosaccharide-bovine serum albumin conjugates, and that, among oligosaccharide ligands tested, preferential binding is to terminal nonreducing N-acetylglucosamine residues of complex type N-linked oligosaccharides. In order to compare the binding specificity of MBP-C, an expression system has been developed for production of a fragment of this protein which contains the COOH-terminal carbohydrate-recognition domain. After radioiodination, the domain has been used to probe natural glycoproteins, neoglycoproteins, and neoglycolipids. Like MBP-A, MBP-C binds several different monosaccharides conjugated to bovine serum albumin, including mannose, fucose, and N-acetylglucosamine, although binding to the last of these is relatively weaker than observed for MBP-A. The results of binding to natural glycoproteins and to neoglycolipids containing oligosaccharides derived from these proteins are most compatible with the interpretation that MBP-C interacts primarily with the trimannosyl core of complex N-linked oligosaccharides, with additional ligands being terminal fucose and perhaps also peripheral mannose residues of high mannose type oligosaccharides. This binding specificity is thus quite distinct from that of MBP-A. The presence of multiple MBPs with distinct binding specificities in preparations derived from serum and liver explains conflicting conclusions which have been reached about carbohydrate recognition by these proteins.     

Isolation and characterization of a mannose-specific endocytosis receptor from rabbit alveolar macrophages.

Rabbit alveolar macrophages express a plasma-membrane receptor that recognizes glycoprotein ligands bearing terminal mannose, fucose or N-acetylglucosamine residues. Macrophage membranes were washed extensively with buffers containing high salt and mannose or EDTA to remove endogenously bound ligand, before Triton X-100 extraction. The extracts were chromatographed on mannose-Sepharose. Elution with mannose, followed by dialysis and a second mannose-Sepharose step with EDTA elution, produced a preparation that migrated as single protein band of Mr 175,000 on SDS/polyacrylamide-gel electrophoresis. The purified protein binds mannose-BSA (bovine serum albumin) with a dissociation constant of 1.9 X 10(-8) M. Ligand binding is Ca2+ and pH-dependent, with maximal binding at neutral pH and low binding below pH 6.0. The binding of 125I-mannose-BSA is inhibited by ligands bearing high-mannose oligosaccharides, such as mannan or beta-glucuronidase, as well as the monosaccharides mannose, fucose and N-acetylglucosamine. Galactose, galactosylated BSA, glucose and mannose 6-phosphate are non-inhibitory. Amino acid compositional analyses indicate that the receptor contains high concentrations of aspartate/asparagine and glutamate/glutamine, and low amounts of methionine. The carbohydrate composition was studied by lectin overlays of electrophoretically transferred receptor, and the results indicate the presence of N-linked complex and O-linked sialylated oligosaccharides. A protein of Mr 175,000 was immunoprecipitated from radio-iodinated macrophage membranes with an antibody generated against purified rabbit lung mannose receptor.     

In vivo recognition of mannosylated proteins by hepatic mannose receptors and mannan-binding protein

In vivo recognition of mannosylated proteins by hepatic mannose receptors and serum mannan-binding protein (MBP) was investigated in mice. After intravenous administration, all three different (111)In-mannosylated proteins were taken up mainly by liver, and uptake was saturated with increasing doses. (111)In-Man-superoxide dismutases and (111)In-Man(12)- and (111)In-Man(16)-BSA had simple dose-dependent pharmacokinetic profiles, whereas other derivatives ((111)In-Man(25)-, -Man(35)-, and -Man(46)-BSA and (111)In-Man-IgGs) showed slow hepatic uptake at <1 mg/kg. Purified MBP experiments in vitro indicated that these derivatives bind to MBP in serum after injection, which interferes with their hepatic uptake. To quantitatively evaluate these recognition properties in vivo, a pharmacokinetic model-based analysis was performed for (111)In-Man-BSAs, estimating some parameters, including the Michaelis-Menten constant of the hepatic uptake and the dissociation constant of MBP, which correlate to the affinity of Man-BSAs for mannose receptors and MBP, respectively. The dissociation constant of Man-BSA and MBP decreased dramatically with increasing density of mannose, but the Michaelis-Menten constant of hepatic uptake of Man-BSA was not so sensitive to the change in density. This suggests that the in vivo recognition of MBP has a stronger cluster effect than that of mannose receptors. Differences obtained here are due to the unique arrangement of carbohydrate recognition domains on each mannose-specific lectin available for mannosylated ligand recognition.     

Involvement of serum mannan binding proteins and mannose receptors in uptake of mannosylated liposomes by macrophages

The roles of serum mannan binding protein (MBP) and the mannose receptor in the cellular uptake of mannosylated liposomes (Man-liposomes) by macrophages were studied. Man-liposomes were prepared by incorporating cholesten-5-yloxy-N-(4-((1-imino-2-beta-D-thiomannosylethyl)amino)butyl)formamide (Man-C4-Chol) into small unilamellar long circulating liposomes consisting of cholesterol (Chol) and distearoyl phosphatidylcholine (DSPC). In the in vitro cellular uptake study with cultured mouse peritoneal macrophages, [(3)H]Man-liposomes were taken up to a great extent, whereas no significant uptake was observed for [(3)H]cholesterol and DSPC liposomes without Man-C4-Chol (Bare-liposomes). The uptake of [(3)H]Man-liposomes was dose- and temperature-dependent and inhibited by an excess of mannosylated bovine serum albumin, suggesting their specific uptake via membrane mannose receptor-mediated endocytosis. Furthermore, it was demonstrated that (111)In-MBP binds strongly to Man-liposomes based on the recognition of Man-C4-Chol and markedly enhanced their uptake by macrophages. These results are supported by confocal laser microscopic images. In addition, in vivo hepatic uptake of (111)In-MBP was enhanced by Man-liposomes. On the other hand, the uptake of Man-liposomes was significantly reduced by preincubation with serum and further with MBP-depleted serum suggesting inhibitory effects of serum proteins such as albumin on mannose receptor-mediated endocytosis. The involvement of serum-type MBP and membrane mannose receptors in the uptake of Man-liposomes is thus suggested.     

Characterization of oligosaccharide ligands expressed on SW1116 cells recognized by mannan-binding protein. A highly fucosylated polylactosamine type N-glycan

Mannan-binding protein (MBP) is a C-type serum lectin and activates complement through the lectin pathway when it binds to ligand sugars such as mannose, N-acetylglucosamine, and fucose on microbes. In addition, the vaccinia virus carrying the human MBP gene was shown to exhibit potent growth inhibitory activity toward human colorectal carcinoma, SW1116, cells in nude mice. We have proposed calling this activity MBP-dependent cell-mediated cytotoxicity (MDCC) (Ma, Y., Uemura, K., Oka, S., Kozutsumi, Y., Kawasaki, N., and Kawasaki, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 371-375). In this study, the MBP ligands on the surface of SW1116 cells were characterized. Initial experiments involving plant lectins and anti-Lewis antibodies as inhibitors of MBP binding to SW1116 cells indicated that fucose plays a crucial role in the interaction. Subsequently, Pronase glycopeptides were prepared from whole cell lysates, and oligosaccharides were liberated by hydrazinolysis. After being tagged by pyridylamination, MBP ligand oligosaccharides were isolated with an MBP affinity column, and then their sequences were determined by mass spectrometry and tandem mass spectrometry after permethylation, in combination with endo-beta-galactosidase digestion and chemical defucosylation. The MBP ligands were shown to be large, multiantennary N-glycans carrying a highly fucosylated polylactosamine type structure. At the nonreducing termini, Le(b)/Le(a) or tandem repeats of the Le(a) structure prevail, a substantial proportion of which are attached via internal Le(x) or N-acetyllactosamine units to the trimannosyl core. The structures characterized are unique and distinct from those of other previously reported tumor-specific carbohydrate antigens. It is concluded that MBP requires clusters of tandem repeats of the Le(b)/Le(a) epitope for recognition.     

Evidence that bovine conglutinin reacts with an early product of C3b degradation, and an improved conglutination assay.

When EAC43b were treated with heated serum in EDTA, reactivity with bovine conglutinin appeared rapidly, even at 0 degrees C, and almost simultaneously with the loss of C3b rosetting capacity. At the time conglutinability first appeared, there was no detectable decrease in I-A or hemolytic C3 activity, and no detectable C3 antigen release from the cells. With prolonged exposure to heated serum in EDTA, I-A (immune adherence) and hemolytic C3 activity were lost. If this exposure was at 37 degrees C, C3 antigen became strongly detectable in the supernatant fluid, and eventually conglutinability was markedly reduced or lost, whereas C3d rosettes were unaffected. We suggest that bovine conglutinin reacts with some early product of C3b degradation, rather than with C3d, and propose that this intermediate be designated C3k. We have developed a semi-quantitative assay for bovine conglutinin, utilizing a Coulter Counter to register the decrease in total particles due to red cell aggregation. By using this method, we have detected conglutination with mouse complement (C) as well as with that from man and the guinea pig.     

Purification of a calcium-activated neutral proteinase from bovine brain

A calcium-activated neutral proteinase (CANP) resolved into three components has been partially purified from bovine brain. The method of isolation has resulted in 22,000, 7,100, and 8,000-fold purification for CANP I, II and III respectively. All three fractions require Ca2+ for activation. The characterization of the purified CANP I has shown that it is activated by 250 microM Ca2+ and the enzyme loses its activity when incubated in the presence of Ca2+ without substrate. Mg2+ is ineffective. The enzyme degrades neurofilament triplet proteins, tubulin and casein efficiently. The myelin basic protein is hydrolyzed after longer incubation. Bovine serum albumin and histones are unaffected. The enzyme is active at pH 5.5 to 9.0 with optimum between pH 7.5 and 8.5. It has a Km of 1.8 X 10(-7) M for the 69,000 dalton neurofilament protein. The enzyme is inhibited by sulphydryl blocking reagents and also by EGTA, leupeptin and E-64c. The SDS-PAGE analysis of the enzyme fractions has shown a major band at 66-68,000 daltons and two minor bands at 60,000 and 48-50,000 daltons for CANP I; a major band at 48-50,000 daltons and a minor band at 30-32,000 daltons for CANP II and a predominant doublet at 30-32,000 daltons with a minor band at 48-50,000 daltons for CANP III. The degradation of neurofilament proteins suggests that the CANP(s) may be involved in the turnover of these proteins.     

Isolation and characterization of a mannan-binding protein from rabbit serum

A binding protein which recognizes mannose and N-acetylglucosamine has been isolated from rabbit serum to apparent homogeneity. The serum binding protein was nearly identical to the mannan-binding protein isolated previously from rabbit liver [Kawasaki, T., Etoh, R. and Yamashina, I. (1978) Biochem. Biophys. Res. Commun. $\text{81}$, 1018–1024] in respect of immunochemical properties and subunit profiles, but could be differentiated from the liver protein in its larger molecular size and inferior sensitivity to monosaccharides as haptenic inhibitors of the binding to ¹²⁵I-mannan. A postulation was made that the plasma was, comparable with the liver, a major locus of mannan-binding protein in the rabbit.     

Identification of N-acetylglucosamine-binding immunoglobulins in chicken egg yolk and serum distinct from the major mannose-binding immunoglobulins.

An N-acetyl-D-glucosamine (GlcNAc)-binding protein of 170 kDa has been isolated from hen serum and egg yolk. Another GlcNAc-binding protein of higher molecular mass was present only in the serum. The 170 kDa protein co-electrophoresed and co-chromatographed in gel filtration with a chicken IgG, and behaved identical to chicken IgG in double immunodiffusion with goat anti-chicken gamma chain antiserum. The sugar-binding hierarchy for the serum and yolk binding proteins, determined with bovine serum albumin neoglycoproteins, was GlcNAc greater than N-acetyl-D-galactosamine greater than glucose = galactose = L-fucose greater than mannose. This hierarchy was unlike any previously reported GlcNAc-binding proteins. The larger serum binding protein component was shown to be an IgM by double immunodiffusion with goat anti-chicken mu chain antiserum. The serum and yolk GlcNAc-binding proteins comprise a unique set of sugar-binding immunoglobulins distinct from the previously reported hen serum and yolk mannose-binding proteins (Wang et al., 1986).     

Apolipoprotein A-I binds to a family of bovine seminal plasma proteins

Bovine seminal plasma contains four similar acidic proteins, previously designated as BSP (bovine seminal plasma)-A1, BSP-A2, BSP-A3, and BSP-30-kDa, that when added to pituitary cell cultures result in the immediate secretion of gonadotropins (follitropin and lutropin). However, when calf or horse serum was included in the culture medium the secretion of gonadotropins was completely prevented. This effect was seen at levels up to 200 micrograms of BSP protein/ml while the presence of more than 200 micrograms of BSP protein/ml in the serum medium continued to release gonadotropins. This could be explained by the presence in the sera of a binding factor to the BSP proteins which prevents their action. This binding factor has been detected in all the sera tested, including human serum, in dot-blot experiments using 125I-labeled BSP-A1, -A2, -A3, or -30-kDa protein. Thus, it was of interest to isolate this binding factor from human serum by affinity chromatography on a column of BSP-A1/-A2-agarose. The purified binding factor was then identified as apolipoprotein A-I (apoA-I) by the following criteria: (a) it has a molecular mass of 27,000 daltons, (b) the amino acid composition is similar to apoA-I, (c) the first 25 residues at the amino-terminal end of this binding factor are identical to apoA-I, and (d) the binding factor cross-reacts in the radioimmunoassay of apoA-I. Furthermore, BSP proteins also bind to purified plasma apoA-I and apoA-I associated with high density lipoprotein. ApoA-I is the major protein of plasma high density lipoprotein and plays an important role in lipid transport and metabolism. Thus, the binding of bovine seminal plasma proteins to apoA-I suggests some physiological significance in lipoprotein function or vice versa.     

Serum mannan binding protein inhibits mannosylated liposome-mediated transfection to macrophages

The effects of serum mannan binding proteins (MBP) in the transfection of plasmid DNA/Man–liposome complex via mannose receptor-mediated endocytosis was studied in vitro using cultured mouse peritoneal macrophages. Plasmid DNA encoding luciferase gene was complexed with cationic mannosylated liposomes (Man–liposomes), composed of cholesten-5-yloxy-N-(4-((1-imino-2-d-thiomannosylethyl)amino)alkyl)formamide (Man-C4-Chol) and dioleoyl phosphatidylethanolamine (DOPE). The transfection efficiency, as well as the binding and uptake of the plasmid DNA/Man–liposome complex, was investigated with or without serum MBP. The in vitro transfection efficiency of the complex was significantly reduced on increasing the amount of serum MBP. In addition, the cellular association of the complex was also reduced. These results indicate that serum MBP specifically binds to the mannose moieties on the complex and suppresses its cellular uptake, resulting in inhibition of the gene transfection in macrophages. Such an interaction is an obstacle to mannose receptor-mediated in vivo gene transfer to mannose receptor-positive cells using mannosylated gene carriers.