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.     

Mannan-binding protein and conglutinin in bovine serum

Conglutinin is a bovine plasma protein which mediates the agglutination of the sensitized erythrocyte-solid phase iC3b complex (conglutination). The serum mannan-binding protein (MBP) is a lectin specific for mannose and N-acetylglucosamine. Since conglutination was shown to be inhibited specifically by N-acetylglucosamine [Leon, M.A. & Yokohari, R. (1964) Science 143, 1327-1328], the possibility was raised that conglutinin might be a bovine serum MBP. The present study, undertaken to solve this problem, revealed that bovine plasma contained an MBP besides conglutinin. These two proteins were very similar in their chemical and physicochemical properties as well as binding specificity. Both bound with high affinity (Kd = 10(-8) M) to glycoproteins terminated with mannose and/or N-acetylglucosamine residues in the presence of calcium, although conglutinin preferred N-acetylglucosamine rather than mannose. They were multimeric proteins of large molecular size (over 1,000,000 daltons, and approximately 600,000 daltons for conglutinin and MBP, respectively) and consisted of a single kind of subunit with molecular weight of around 45,000. The MBP was shown to have a collagen-like structure in the molecule, as was recently reported for conglutinin [Davis, A.E., III & Lachmann, P.J. (1984) Biochemistry 23, 2139-2144]. Despite these similarities, the MBP and conglutinin were immunochemically distinct, and the MBP did not show any conglutination activity.     

Structure of the complex oligosaccharides of fetuin.

The complete structure of the complex oligosaccharides of fetuin has been established. The three fractions of complex oligosaccharide which were isolated by ion exchange chromatography following pronase digestion (F-I, F-II, and F-III) had identical molar ratios of sialic acid (Sia), galactose, mannose, and N-acetylglucosamine of 3:3:3:5. A combination of methylation analyses, Smith periodate degradations, and endoglycosidase and exoglycosidase digestions were utilized to establish the structure which is proposed to be: (see article of journal). Features of this structure not previously established include the presence of 2 residues of alpha2,3- and 1 residue of alpha2,6-linked sialic acid and their location relative to the mannose branch points. Also unusual is the presence of an alpha-linked branch mannose with substituents at positions 2 and 4 which is in turn linked to position 6 of the beta-linked, branch mannose. These features result in unexpected resistance to specific exoglycosidases.     

Purification and some properties of a novel alpha-L-fucosidase capable of acting on alpha-(1----6)-L-fucosidic linkages from Bacillus circulans M28

Two types of alpha-L-fucosidase (F-I and F-II), that differ in substrate specificity, were produced in the culture fluid by Bacillus circulans isolated from soil when the bacterium was cultivated on medium containing porcine gastric mucin. F-I was able to cleave the alpha-(1----2), alpha-(1----3), and alpha-(1----4)-L-fucosidic linkages in various oligosaccharides and glycoproteins, but not p-nitrophenyl alpha-L-fucoside, as previously reported [Y. Tsuji et al. (1990) J. Biochem. 107, 324-330]. F-II was purified from the culture fluid obtained with glucose medium by ammonium sulfate fractionation and various subsequent column chromatographies. The purified enzyme was found to be homogeneous on PAGE and its molecular weight was estimated to be approximately 250,000. The maximal activity was observed between pH 6.0 to 7.0, the stable pH range being 6.0 to 8.5. The enzyme specifically cleaved alpha-L-fucosidic bonds in low molecular weight substrates. The enzyme cleaved not only p-nitrophenyl alpha-L-fucoside, but also 2-fucosyllactose and 3-fucosyllactose. The enzyme was also able to act on the alpha-(1----6)-L-fucosidic linkages to N-acetylglucosamine in 6-O-alpha-L-fucopyranosyl-N-acetylglucosamine, and bi- and tetra-antennary oligosaccharides derived from porcine pancreatic lipase, which were not hydrolyzed by F-I.     

Isolation of two endo-beta-N-acetylglucosaminidases from fig latex

Two endo-beta-N-acetylglucosaminidases (mannosyl-glycoprotein 1,4-N-acetamidodeoxy-beta-D-glycohydrolase, EC 3.2.1.96) (type F-I and type F-II) have been isolated from fig latex. At pH 7.0, type F-1 was retained by the DEAE-Sephadex A-50 column, whereas type F-II was not adsorbed by the column. The optimum pH of type F-I was found to be pH 5.9 and type F-II, pH 5.4. Type F-I enzyme hydrolyzes the tri-mannosyl derivatives di-N-acetylglucosaminylasparagine faster than the penta- or hexa-mannosyl compounds. Type F-II hydrolyzes the penta- and hexa-mannosyl derivatives, but not the tri-mannosyl compound.     

Multiple molecular forms of mouse liver arginase

Hepatic arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is an oligomer composed of three or four subunits. The present studies indicate heterogeneity in the size and charge of arginase subunits in mouse liver. Two types of arginase subunits with molecular weights of approximately 35,000 and 38,000 have been found. These two subunits are detected in liver cytosol or in purified preparations of arginase after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Two dimensional SDS-PAGE revealed multiple ionic forms of arginase for both the 35,000 and 38,000 subunits; the subunits contain basic proteins (pI range 7.8-9.1) and acidic proteins (pI range 5.8-6.4). Limited proteolysis by trypsin eliminated the molecular weight differences between the subunits without substantially affecting either their isoelectric points or activity. Comparative peptide maps and amino acid analyses of the 35,000- and 38,000-Da subunits showed that they were very similar. The data indicate that a neutral peptide (approx 3000 Da) is responsible for the differences in subunit molecular weight and that the multiple sized and charged forms are variants of the same protein.     

The binding of fucose-containing glycoproteins by hepatic lectins. Purification of a fucose-binding lectin from rat liver

A lectin with a high affinity for binding ligands through fucose residues has been purified to homogeneity from rat liver. Affinity chromatography of the lectin on fucosyl-bovine serum albumin-agarose is the key step in the purification. Contaminating amounts of a previously described lectin that binds mannose and N-acetylglucosamine are removed from the fucose-binding lectin by either immunoadsorption on anti-mannose/N-acetylglucosamine lectin IgG-agarose or by specific elution of the fucose-binding lectin from fucosyl-bovine serum albumin-agarose. The pure fucose-binding lectin contains two polypeptide subunits with molecular weights of 88,000 and 77,000, respectively, as judged by gel electrophoresis. Peptide maps of the subunits, however, show that they are very similar structurally. In addition, peptide maps show that the fucose lectin is structurally distinct from other rat hepatic lectins. This is supported by the lack of cross-reaction among the different rat liver lectins and their specific antibodies and the inability of specific antibodies to the mannose/N-acetylglucosamine lectin to inhibit the binding of fucosyl-bovine serum albumin by the fucose lectin.     

Metabolism of Cytokinin : DEPHOSPHORYLATION OF CYTOKININ RIBONUCLEOTIDE BY 5'-NUCLEOTIDASES FROM WHEAT GERM CYTOSOL

Two forms (F-I and F-II) of 5′-nucleotidases (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) which catalyze the dephosphorylation of N⁶-(Δ²-isopentenyl)adenosine 5′-monophosphate and AMP to form the corresponding nucleosides were partially purified from the cytosol of wheat (Triticum aestivum) germ. Both the F-I (molecular weight, 57,000) and F-II (molecular weight, 110,000) 5′-nucleotidases dephosphorylate the ribonucleotides at an optimum pH of 7. The K_m values for the cytokinin nucleotide are 3.5 micromolar (F-I enzyme) and 12.8 micromolar (F-II enzyme) in 100 millimolar Tris-maleate buffer (pH 7) at 37 C. The F-I enzyme is less rapidly inactivated by heating than is the F-II enzyme. Both nucleotidases hydrolyze purine ribonucleoside 5′-phosphates, AMP being the preferred substrate. N⁶-(Δ²-isopentenyl)Adenosine 5′-monophosphate is hydrolyzed at a rate 72 and 86% that of AMP by the F-I and F-II nucleotides, respectively. Phenylphosphate and 3′-AMP are not substrates for the enzymes. It is proposed that dephosphorylation of cytokinin nucleotide by cytosol 5′-nucleotidases may play an important role in regulating levels of “active cytokinin” in plant cells.     

Primary structure of rat liver mannan-binding protein deduced from its cDNA sequence

cDNA clones encoding rat liver mannan-binding protein (MBP), a lectin specific for mannose and N-acetylglucosamine, were isolated from a rat liver cDNA library carried in lambda gt 11, by screening with affinity purified polyclonal rabbit anti-rat liver MBP antibodies. The nucleotide sequence of the cDNA determined by the dideoxy method revealed the complete amino acid sequence of the MBP (226 residues). The NH2-terminal residue of the MBP, glutamic acid, was preceded by a predominantly hydrophobic stretch of 18 amino acids, which was assumed to be a signal peptide. Near the NH2-terminal, there was a collagen-like domain, which consisted of 19 repeats of the sequence Gly-X-Y. Here, X and Y were frequently proline and lysine. Three proline and lysine residues were hydroxylated, and one of the latter appeared to link to galactose. Computer analysis of several lectins for sequence homology suggested that the COOH-terminal quarter of the MBP is associated with the calcium binding as well as carbohydrate recognition.     

Isolation and chemical characterization of algal polysaccharides from the green seaweed Ulva clathrata (Roth) C. Agardh

In order to obtain information on the content and composition of the water-soluble polysaccharides from Ulva clathrata, an extraction at 60°C, in different media, was performed: water, EDTA and HCl (F-I), each followed by a sequential extraction in NaOH 0.1 M (F-II). The extracts were recovered and analyzed for total carbohydrates, proteins, rhamnose, uronic acids and sulfate content. Differences were obtained in the yield and composition in both fractions of the different media (F-I and F-II). Higher yields resulted in the first fraction on all media. HCl extraction was the best in both fractions (14.83 ± 1.5% and 5.96 ± 1.1%, F-I and F-II, respectively). In all cases, F-I was more sulfated ranging from 27.87% to 35.8% and higher in rhamnose content, whereas F-II had higher protein and slightly higher uronic acid content. FTIR spectra showed that soluble polysaccharides from the green seaweed U. clathrata are sulfated polysaccharides, similar to ulvan obtained from other Ulva species and confirmed by the ¹ H-NMR spectrum, where the characteristic signal for the deoxy sugar (rhamnose) is present.     

Identification of two lipid binding proteins from liver of Gallus domesticus

1. Two low molecular weight (approximately 14,000 Da) proteins exhibiting lipid binding activity were purified from liver cytosol and identified as non-specific lipid binding protein (ns-LTP) and fatty acid binding protein (L-FABP). 2. Ligand binding assays indicated that ns-LTP exhibited greater binding activity for cholesterol and little binding of fatty acids. Conversely, L-FABP had higher relative binding activity for fatty acids but did not bind cholesterol. 3. Amino acid composition and pI data supported the identification of the chicken liver lipid binding proteins as L-FABP and ns-LTP. 4. Polyclonal antisera was prepared against each of the liver lipid binding proteins and monospecificity verified using Western blot analysis.     

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

A membrane protein which binds mannan has been isolated from rabbit liver by affinity chromatography. Upon polyacrylamide gel electrophoresis, a single major band was recovered with an estimated molecular weight of 31,000. When assayed as inhibitors, N-acetylmannosamine, N-acetylglucosamine, and mannose were potent inhibitors of mannan binding; N-acetylgalactosamine and mannose-6-phosphate were inert. Glycoproteins with terminal N-acetylglucosamine and/or mannose residues which are cleared rapidly from the circulation by the liver were the most potent inhibitors. On the basis of these results, it is proposed that the mannan-binding protein is the principle mediating the hepatic uptake of glycoproteins with terminal N-acetylglucosamine and/or mannose residues.     

Purification and characterization of acid phosphatase in rat liver lysosomal contents

Acid phosphatase in rat liver lysosomal contents, C-APase I, was purified about 5,700-fold over the homogenate with 8.0% recovery, to apparent homogeneity as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The purification procedures included; preparation of crude lysosomal contents, DEAE-Sephacel ion exchange chromatography, hydroxylapatite chromatography, and gel filtration with Sephacryl S-300. The enzyme is composed of three identical subunits with an apparent molecular weight of 48K. The enzyme contains about 11% carbohydrate and the carbohydrate moiety was composed of mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine in a molar ratio of 20:3:11:1. Sialic acid was not detected in the enzyme. Antisera against the purified C-APase I were raised in goat and the C-APase I was rapidly purified with high yield (10%) by using the specific antibodies coupled to Sepharose 6B.     

Cloning and characterization of a novel mannose-binding protein of Acanthamoeba

Acanthamoebae produce a painful, blinding infection of the cornea. The mannose-binding protein (MBP) of Acanthamoeba is thought to play a key role in the pathogenesis of the infection by mediating the adhesion of parasites to the host cells. We describe here the isolation and molecular cloning of Acanthamoeba MBP. The MBP was isolated by chromatography on the mannose affinity gel. Gel filtration experiments revealed that the Acanthamoeba lectin is a approximately 400-kDa protein that is constituted of multiple 130-kDa subunits. Cloning and sequencing experiments indicated that the Acanthamoeba MBP gene is composed of 6 exons and 5 introns that span 3.6 kb of the amoeba genome and that MBP cDNA codes for a precursor protein of 833 amino acids. That the cloned cDNA encodes authentic MBP was demonstrated by showing that: (i). recombinant MBP possesses mannose binding activity, and (ii). polyclonal antibodies prepared against Acanthamoeba MBP bound to the recombinant protein. Sequence analysis revealed that the MBP contains a large N-terminal extracellular domain, a transmembrane domain, and a short C-terminal cytoplasmic domain. Despite extensive BLAST searches using the MBP sequence, no significant matches were retrieved. The most striking feature of the Acanthamoeba MBP sequence is the presence of a cysteine-rich region containing 14 CXCXC motifs within the extracellular domain. In summary, we have isolated, cloned, and characterized a novel MBP from Acanthamoeba. Because the presence of antibodies to MBP in tears provides protection against infection, the availability of the MBP cDNA sequence and rMBP should help develop: (i). a tear-based test to identify individuals who are at risk of developing the keratitis and (ii). strategies to immunize high-risk individuals.     

Mannan-binding protein in lymphoid tissues of rats

A binding protein which recognizes mannose and N-acetylglucosamine was isolated from mesenteric lymph nodes of rats by affinity chromatography. The isolated binding protein shares come common properties with liver mannan-binding protein: requirement of Ca2+ for the binding and high affinity for mannan. However, these two proteins were distinguishable by their antigenicity and their binding affinity for mannosamine.     

Isolation and partial characterization of a calcium-dependent lectin (chiletin) from the haemolymph of the flat oyster, Ostrea chilensis

A calcium-dependent lectin (chiletin) was isolated from oyster haemolymph by mannose elution from Sepharose CL-6B followed by anion exchange chromatography. Chiletin was predominantly composed of 12 and 24 kDa bands when examined with SDS-PAGE under reducing and non-reducing conditions, respectively. Larger molecular weight bands of 36 and 50 kDa were also variably present under reducing conditions. The NH2-terminal sequence of the 24 kDa band was determined and was not homologous to any known protein from the databases searched. Isolated chiletin was composed of multiple isomers approximately 12 kDa in size and ranging in pI from 5.2 to 6.0. Rabbit antiserum was raised to a synthetic peptide coupled to keyhole limpet hemocyanin and the size of the chiletin subunits was confirmed by Western blot. Two and five different conformational aggregates of chiletin were resolved in oyster haemolymph using size exclusion chromatography in 8 M urea and PBS, respectively. The largest aggregate obtained from size exclusion in 8 M urea was estimated to be greater than 640 kDa. The ability of whole haemolymph and isolated chiletin to agglutinate sheep red blood cells was inhibited by galactose and mannose. Chiletin was identified by immunohistochemistry to be most consistently present in the auricle, followed by the digestive gland, however staining was seen sporadically in haemocytes, gastrointestinal epithelium and interstitial connective tissue cells.     

Isolation and characterization of cyanogen bromide fragments and a glycopeptide from the Dolichos biflorus lectin.

The 110000 molecular weight Dolichos biflorus lectin is a glycoprotein composed of four subunits of approximately 27000 molecular weight with one methionine residue per subunit (Carter and Etzler, 1975b). Cyanogen bromide cleavage of the lectin yielded two fragments with approximate molecular weights of 15000 and 12000 as determined by electrophoresis on sodium dodecyl sulfate gels. Only the 15000 molecular weight fragment stained for carbohydrate with the periodic acid-Schiff stain. The two fragments were isolated, and their amino acid compositions were determined. The 15000 molecular weight fragment was identified as the amino terminal segment of the lectin subunits by NH2-terminal amino acid analysis. A glycopeptide with a minimum molecular weight of 1100 was isolated from the lectin by exhaustive Pronase digestion. Complete acid hydrolysis of the glycopeptide yielded aspartic acid, mannose, and N-acetylglucosamine in the ratio of 1:4-5:1-2. Partial acid hydrolysis of the glycopeptide produced a component which had an identical mobility with commercial N-acetylglucosaminylasparagine in high voltage paper electrophoresis. The data indicate that the carbohydrate unit of the lectin is bound to the amino terminal half of the subunits by a glycosylamine linkage between N-acetylglucosamine and asparagine.     

Purification and characterization of glutathione transferases from the sea bass (Dicentrarchus labrax) liver

Two forms of glutathione transferase were purified from liver cytosol of the sea bass (Dicentrarchus labrax) by GSH-Sepharose affinity chromatography followed by chromatofocusing. The major enzyme (DL-GST-6.7; 75% of total activity bound to the column) has a pI value of 6.7 and is composed of two subunits of apparent molecular mass 26.5 kDa. The minor enzyme (DL-GST-8.2; 25% of total activity bound to the column) has a pI value of 8.2 and is composed of two subunits of molecular mass 23.5 kDa. Both isoenzymes appear to have blocked N-terminal. The purified proteins were characterized with respect to substrate specificity, CD spectra, TNS binding properties (with 2-toluidinylnaphthalene 6-sulfonate), and immunological reactivity. Partial internal amino acid sequence was also determined for each isoenzyme. The results obtained suggest that DL-GST-6.7 and DL-GST8.2 are novel GSTs belonging, respectively, to theta and alpha classes.     

Cardiac fatty acid-binding proteins. Isolation and characterization of the mitochondrial fatty acid-binding protein and its structural relationship with the cytosolic isoforms

In the course of our studies on the structural diversity of the isoforms of cardiac fatty acid-binding proteins (cFABPs), a cardiac-type FABP from the matrix of bovine heart mitochondria was purified to homogeneity and obtained as a single 15-kDa protein with an isoelectric point of 4.9. The primary structures of this protein and of the two isoforms isolated from the cytosol (pI4.9-cFABP and pI 5.1-cFABP) were investigated by means of plasma desorption mass spectrometry and sequencing of peptides. All three proteins are amino-terminally blocked with an acetyl group and shown to be colinear with the sequence deduced from a cDNA clone for bovine heart fatty acid-binding protein (Billich, S., Wissel, T., Kratzin, H., Hahn, U., Hagenhoff, B., Lezius, A. G., and Spener, F. (1988) Eur. J. Biochem. 175, 549-556) except for the residue at position 98. This residue is demonstrated to be the molecular origin of bovine cFABP isoforms since pI 5.1-cFABP contains Asn98 in accordance with the sequence derived from the cDNA, whereas in pI 4.9-cFABP, this position is occupied by Asp98. Moreover, mitochondrial FABP is identical to pI 4.9-cFABP. Molecular masses of pI 4.9-cFABP (14,679 +/- 10 Da) and pI 5.1-cFABP (14,678 +/- 20 Da) determined by plasma desorption mass spectrometry coincide with that calculated from the cDNA (14,673 Da). Hence, residues linked to these proteins by posttranslational modification are not present, and the Asn-Asp exchange is the sole origin of heterogeneity of mitochondrial and cytosolic fatty acid-binding proteins from bovine heart.     

Isolation and characterization of endogenous ligands for liver mannan-binding protein

Endogenous ligands for the hepatic lectin which is specific for mannose and N-acetylglucosamine (mannan-binding protein, MBP) were isolated from rat liver rough microsomes and primary cultured hepatocytes by affinity chromatography on an immobilized MBP column. Western blotting using specific antisera revealed that serum glycoproteins, alpha 1-macroglobulin, alpha 1-antitrypsin, and alpha 1-acid glycoprotein, and a lysosomal enzyme, beta-glucuronidase were the major constituents of the endogenous ligands. These endogenous ligands consisted of high mannose-type oligosaccharides of Man9GlcNAc2 and Man8GlcNAc2, and had rapid turnover rates with an average half-life of 45 min, indicating that they were mainly composed of biosynthetic intermediates of glycoproteins. In view of the identification of the endogenous ligands as the biosynthetic intermediates of glycoproteins, the possible functions of the intracellular lectin are discussed in relation to the intracellular transport of glycoproteins.