Mannose-specific isolectins with different hemagglutinating potencies isolated from Chinese daffodil (Narcissus tazetta var. chinensis) leaves

Three mannose-specific lectins exhibiting considerable similarities in NH₂-terminal amino acid sequence were isolated from leaves of the Chinese daffodil Narcissus tazetta (Family Amaryllidaceae). The purification protocol involved extraction with an aqueous buffer, anion exchange chromatography on DEAE-cellulose using stepwise elution with increasing salt concentrations, affinity chromatography on mannose-agarose, and FPLC-gel filtration on Superose 12. From the peak unadsorbed on DEAE-cellulose, and two peaks adsorbed on the ion exchanger and eluted respectively with 0.2 M Tris-HCl buffer and 0.5 M NaCl, were prepared fractions which yielded isolectins 1, 2, and 3 after adsorption on mannose-agarose and FPLC-gel filtraton. All three isolectins were homodimers with a molecular weight of 26 kDa. The lectin unadsorbed on DEAE-cellulose had the lowest, while the most strongly adsorbed lectin had the highest hemagglutinating activity.     

A synthetic peptide derived from p34cdc2 is a specific and efficient substrate of src-family tyrosine kinases.

A peptide derived from p34cdc2, cdc2(6-20)NH2 with the amino acid sequence of KVEKIGEGTYGVVYK-amide, was found to be a specific and efficient substrate for a pp60c-src-related protein tyrosine kinase from bovine spleen (STK). Glu-12 and Thr-14 were identified to be substrate specificity determinants in this peptide (Cheng, H.-C., Litwin, C. M. E., Hwang, D. M., and Wang, J. H. (1991) J. Biol. Chem. 266, 17919-17925). In this study, we demonstrated the presence of cdc2(6-20)NH2 peptide tyrosine kinase activity in the membrane fractions of bovine brain, spleen, thymus, lung, liver, and kidney. Hydroxylapatite column chromatography of thymus membrane extract revealed four protein tyrosine kinases, TK-I, TK-II, TK-III, and TK-IV, with different relative activities toward cdc2(6-20)NH2 and a general tyrosine kinase substrate, poly(Glu/Tyr). Only TK-I and TK-II showed significant activity toward cdc2(6-20)NH2, they were suggested as belonging to the src-family by virtue of their cross-reactivity with an antibody against a synthetic peptide corresponding to a conserved sequence of src-family kinases. Further immunological characterization using antibodies specific to individual src-related protein tyrosine kinases suggested that TK-I, TK-II, and STK are bovine homologs of p56lck, p55fyn, and p56lyn, respectively. Substrate specificity and kinetic characterization of src-family tyrosine kinases including human platelet pp60c-src, bovine p56lyn, p56lck, and p55fyn, as well as several non-src-related tyrosine kinases including epidermal growth factor receptor, p43v-abl, TK-III, and TK-IV showed that all the src-family tyrosine kinases but none of the other kinases displayed efficient cdc2(6-20)NH2 phosphorylation. In all cases, the high efficiency of cdc2(6-20)NH2 peptide phosphorylation could be markedly attenuated when Glu-12 and Thr-14 of the peptide were substituted, respectively, by valine and serine.     

Purification and characterization of two lectins from Aloe arborescens Mill.

Two lectins have been isolated from leaves of Aloe arborescens Mill by salt precipitation, pH-dependent fractionation and gel filtration. One lectin (P-2) has a molecular weight of approximately 18,000, consists of two subunits (alphabeta) and contains more than 18% by weight of neutral carbohydrate. The smaller subunit (alpha) has a molecular weight of approximately 7,500 and the larger subunit (beta) a molecular weight of approximately 10,500. The other lectin (S-1) has a molecular weight of approximately 24,000, consists of two subunits (gamma2) with a molecular weight of approximately 12,000 and contains more than 50% by weight of neutral carbohydrate. An interesting feature of the amino acid compositions of these lectins is the high proportion of acidic amino acids, such as aspartic acid and glutamic acid, and the low proportion of methionine and histidine. S-1 has a strong hemagglutinating activity. On the other hand, P-2 has not only hemagglutinating activity but also mitogenic activity on lymphocytes, precipitate-forming reactivity with serum proteins, one of which is alpha2-macroglobulin, and complement C3 activating activity via the alternate pathway.     

Study of the primary structures of the peptide core of bovine estrus cervical mucin. Possible existence of small similar subunits

Two populations of tryptic peptides were isolated from bovine estrus cervical mucin (BCM). One contained all the carbohydrate, and was rich in threonine and serine. These glycopeptides had, like the whole mucin, alanine as their NH2-terminal residues. Their COOH-terminal residues were arginine. The second population of peptides was rich in carboxylic amino acids, contained two cysteinyl residues, and had, like the whole mucin, leucine as COOH-terminal residues. Their NH2-terminal residues were aspartic acid. The sum of the residues of one glycopeptide plus one cysteinyl-containing peptide corresponded to the number of residues constituting a putative subunit of BCM. The amino acid sequence of the major cysteinyl peptide was determined. A cluster of hydrophobic residues was found in the COOH-terminal region. The amino acid sequences of two of the glycopeptides were found identical up to the 22nd residue. The small number of tryptic peptides, as well as the large amount of NH2- and COOH-terminal amino acids found in BCM indicate that this glycoprotein is made up of similar subunits with a molecular weight of about 22,000, one of the glycopeptides representing the NH2-terminal part, and one of the cysteinyl peptides, the COOH-terminal part. However, the existence of these subunits was not confirmed by ultracentrifugation of BCM in dithiothreitol and sodium dodecyl sulfate. BCM was polydisperse and had a mean molecular weight of 507,000.     

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.     

Primary structure of human fibrinogen and fibrin. Structural studies on NH2-terminal part of B beta chain.

The cyanogen bromide fragment, N-DSK, containing the NH2-terminal portions of the three chains of fibrinogen, was found to exist in dimeric and polymeric forms. These different forms gave rise to identical chain fragments on reduction and alkylation. The B beta chain of N-DSK from fibrinogen and the beta chain of N-DSK from fibrin were isolated and characterized. The B beta chain fragment has a blocked NH2-terminal residue, and fibrinopeptide B is released on digestion with thrombin. The beta chain fragment has glycine as NH2-terminal residue. The molecular weight of the B beta chain fragment is 12200 as determined by ultracentrifugal analysis. Gel electrophoresis in sodium dodecyl sulphate gave the molecular weights of 14000 and 13000 for the B beta chain and beta chain fragments, respectively. The NH2-terminal B beta chain fragment consists of 118 amino acid residues and the beta chain fragment of 104 residues. The amino acid sequence of beta chain fragment is identical to B beta chain fragment except for the fibrinopeptide B portion. The isolation of a B beta-related fragment (B beta +), with a molecular weight of 30000, is also reported. The presence of B beta + was explained on the basis of incomplete cleavage at the Met-118 residue during treatment with cyanogen bromide. Some functional aspects of the B beta chain fragment are discussed.     

Occurrence of highly yielded lectins homologous within the genus Eucheuma

We previously reported that the red alga Eucheuma serra contains large amounts of mitogenic isolectins (ESA-1 and ESA-2), the hemagglutinating activities of which were strongly inhibited by glycoproteins bearing high mannose-type N-glycans. We therefore further examined two other species, E. amakusaensis and E. cottonii. Several lectins were isolated easily by a combination of extraction with aqueous ethanol, precipitation with cold ethanol, gel filtration, and ion exchange chromatography from both species, respectively. The purified lectins were designated as EAA-1, EAA-2, EAA-3, ECA-1 and ECA-2 after the specific names of both algae. The yields of EAAs and ECAs were as high as 2.8 and 2.7 mg g⁻¹ of dry tissue, respectively, indicating that both species would also be good sources for high lectin yields. The five purified lectins shared the same properties in hemagglutinating activity, mitogenic activity, and hemagglutination-inhibition test in which glycoproteins bearing high mannose-type N-glycans were the most inhibitory. They also had almost identical molecular weight and 20 N-terminal amino acid sequence to each other and to those of ESAs, and only differed in the isoelectric point, indicating that they are isolectins to each other. The study thus demonstrated that several species of Eucheuma contain high yields of lectins homologous between species, suggesting that the genus as a whole may be considered as a valuable source of lectin proteins. This revised version was published online in July 2006 with corrections to the Cover Date.     

Primary structure of human fibrinogen and fibrin. Isolation and partial characterization of chains of fragment D.

Fragment D has been isolated as an apparently single molecular weight species (molecular weight about 100,000) from plasmin digests of humman fibrinogen, using a combination of affinity chromatography on insolubilized "fibrin monomer" and gel filtration. This fragment consists of three chains with molecular weights of 15,000 (Dbeta), 42,500 (Dgamma1) or 39,500 (Dgamma2), and 14,000 (Dalpha) held together by disulfide bonds. The S-carboxymethyl derivatives of the chains have been separated by gel filtration and ion exchange chromatography, and their identity has been confirmed by peptide mapping and immunological analysis. The chain with a molecular weight of 45,000 is a fragment of the Bbeta chain of fibrinogen. The chain derived from the gamma chain of fibrinogen occurred in two molecular forms having molecular weight 42,500 and 39,500. The chain derivative with molecular weight 14,000 is most likely derived from the Aalpha chain of fibrinogen. The chains were characterized by NH2-terminal sequence analysis, amino acid composition, and carbohydrate staining. The two molecular analysis, amino acid composition, and carbohydrate staining. The two molecular forms of the gamma chain appeared to be identical except for an NH2-terminal peptide extension of 23 amino acid residues in the longer chain. The latter has sequences in common with the COOH-terminal part of the gamma chain of the NH2-terminal disulfide knot (BROMBACK, B., BRONDAHL, N. J., HESSEL, B., IWANAGA, S., and WALLEN, P. (1973) J. Biol. Chem. 248, 5806-5820); its NH2-terminal residue being Ala-63 of the gamma chain of fibrinogen.     

Physical-chemical characterization and carbohydrate-binding activity of the A and B subunits of the Bandeiraea simplificolia I isolectins.

Bandeiraea simplicifolia I plant seed isolectins comprise a family of tetrameric alpha-D-galactopyranosyl-binding glycoproteins composed of various combinations of teo different kinds of subunits designated A and B. Subtypes of the A (Aa, Ab, Ac, Ad, and Ae) and B (Ba, Bb, Bc, Bd, and Be) subunits were demotypes varies from seed to seed (e.g., some seeds contain only B subunits, others only A subunits), subtypes Ac and Bc predominate in a natural mixture of the isolectins. Two-dimensional agar gel diffusion studies indicate that, in addition to common structural features, each subunit contains its own distinct antigenic determinants. Although the A and B subunits have closely similar amino acid compositions, they differ markedly in one respect: the B subunit has one methionine residue whereas the A subunit contains no methionine. The neutral carbohydrate content of both subunits is identical. The ability of biopolymers and synthetic glycoproteins to precipitate A4 and B4, as well as the capacity of sugars and oligosaccharides to inhibit precipitate formation, was examined. On the basis of these studies, it is suggested that hydrogen bonding occurs between the hydrogen atoms of the C-3 and C-4 hydroxyl groups of alpha-D-GalNAcp and alpha-D-galp units and the A and B subunits, respectively.     

Purification and characterization of a 1,2-dihydroxynaphthalene dioxygenase from a bacterium that degrades naphthalenesulfonic acids.

1,2-Dihydroxynaphthalene dioxygenase was purified to homogeneity from a bacterium that degrades naphthalenesulfonic acids (strain BN6). The enzyme requires Fe2+ for maximal activity and consists of eight identical subunits with a molecular weight of about 33,000. Analysis of the NH2-terminal amino acid sequence revealed a high degree of homology (22 of 29 amino acids) with the NH2-terminal amino acid sequence of 2,3-dihydroxybiphenyl dioxygenase from strain Pseudomonas paucimobilis Q1. 1,2-Dihydroxynaphthalene dioxygenase from strain BN6 shows a wide substrate specificity and also cleaves 5-, 6-, and 7-hydroxy-1,2-dihydroxynaphthalene, 2,3- and 3,4-dihydroxybiphenyl, catechol, and 3-methyl- and 4-methylcatechol. Similar activities against the hydroxy-1,2-dihydroxynaphthalenes were also found in cell extracts from naphthalene-degrading bacteria.     

Isolation and Characterization of Isolectins from Talisia esculenta Seeds

Four isolectins (TEL-I, TEL-II, TEL-III and TEL-IV) were isolated from seeds of Talisia esculenta by reverse-phase high-performance liquid chromatography. RP-HPLC was performed on a u-Bondapack C18 column (0.78 cm × 30 cm) (Waters 991-PDA system) at room temperature. Rechromatography of the four fractions on a C18 column under the same conditions yielded lectins with two dissimilar subunits (M_r 20 kDa and 40 kDa) bound noncovalently. The isolectins showed very similar characteristics, such as molecular masses, N-terminal sequences, and hemagglutinating activity, but differed in their isoelectric points and in inhibition by carbohydrates.     

Purification and characterization of the trifunctional beta-subunit of anthranilate synthase from Neurospora crassa

The trifunctional beta-subunit of anthranilate synthase complex of Neurospora crassa has been purified from a mutant which produces no detectable alpha-subunit. The isolated beta-subunit appeared to be a highly asymmetric dimer with a s20,w of 7.35 and an apparent molecular weight of 200,000 as determined by gel filtration on Sephacryl S-300 compared with a monomer molecular weight of approximately 84,000 Da as determined by sodium dodecyl sulfate-gel electrophoresis. The purified subunit was cleaved by elastase, trypsin, or chymotrypsin into fragments which retained the three enzyme activities. After elastase digestion, two active fragments were separated by gel filtration and ion exchange chromatography. A 30,000-Da fragment, which behaved as a monomer on gel filtration, interacted with free alpha-subunit to produce glutamine-dependent anthranilate synthase activity. A second 56,000-Da fragment, which behaved as an asymmetric dimer (apparent molecular weight 140,000) on gel filtration, retained both N-(5'-phosphoribosyl)anthranilate isomerase and indole-3-glycerol phosphate synthase activity. The failure to detect an NH2-terminal amino acid residue on either the intact beta-subunit or the 30,000-Da complementing fragment, while the 56,000-Da fragment possessed an NH2-terminal histidine residue, indicated that the complementing fragment was derived from the NH2-terminal sequence of the beta-subunit.     

Similar structures in gamma-carboxymuconolactone decarboxylase and beta-ketoadipate succinyl coenzyme A transferase.

gamma-Carboxymuconolactone decarboxylase (EC 4.1.1.44) and beta-ketoadipate succinyl coenzyme A transferase (EC 2.8.3.6) mediate different steps in the beta-ketoadipate pathway. Antisera prepared against the Pseudomonas putida transferase cross-reacted immunologically with the decarboxylase from the same organism. The transferase is formed by association of two nonidentical protein subunits. The NH2-terminal amino acid sequences of the two nonidentical transferase subunits resembled each other and also were similar to the NH2-terminal amino acid sequence of the decarboxylase.     

Characterization of the subunits of beta-conglycinin

Four subunits of beta-conglycinin were purified from soybean cultivar CX 635-1-1-1, and were designated alpha, alpha', beta, and beta' in accordance with nomenclature proposed by Thanh and Shibasaki [(1977) Biochim. Biophys. Acta 490, 370-384]. Of these subunits, beta' has not previously been reported or characterized. Consistent with the low levels of methionine in these proteins, cyanogen bromide cleavage of alpha', alpha, and beta' subunits produced only a few fragments. The beta subunit contains no methionine and was not cleaved by cyanogen bromide. The NH2-terminal amino acid sequences of the alpha and alpha' subunits are homologous, and each has valine at its amino terminus. The beta subunit has a very different NH2-terminal sequence from those of the alpha and alpha' subunits, and has leucine at its amino terminus. The NH2-terminal sequence of the beta' subunit could not be determined, as it appeared to be blocked to Edman degradation. Although alpha and alpha' subunits have similar NH2-terminal sequences, they differ in the number of methionine residues and so yielded different numbers of cyanogen bromide fragments. Two cyanogen bromide fragments (CB-1 and CB-2) were purified from the alpha subunit. CB-1 originated from the NH2-terminal end of the subunit. The amino acid sequence of CB-2 was identical to that predicted from the nucleotide sequence of cDNA clone pB36. The insert in pB36 encoded 216 amino acids from the COOH-terminal end of the alpha subunit and contained a 138-bp trailer sequence which was followed by a poly-(A) tail. Maps showing the relative positions of methionine residues and carbohydrate moieties in the alpha and alpha' subunits were drawn, based on primary sequence data, and the size and carbohydrate content of the CNBr fragments derived from the subunits.     

Specificity of binding of NH2-terminal residue of proteins to ubiquitin-protein ligase. Use of amino acid derivatives to characterize specific binding sites

Previous studies have indicated that at least part of the selection of proteins for degradation takes place at a binding site on ubiquitin-protein ligase, to which the protein substrate is bound prior to ligation to ubiquitin. It was also shown that proteins with free NH2-terminal alpha-NH2 groups bind better to this site than proteins with blocked NH2 termini (Hershko, A., Heller, H., Eytan, E., and Reiss, Y. (1986) J. Biol. Chem. 261, 11992-11999). In the present study, we used simple derivatives of amino acids, such as methyl esters, hydroxamates, or dipeptides, to examine the question of whether the protein binding site of the ligase is able to distinguish between different NH2-terminal residues of proteins. Based on specific patterns of inhibition of the binding to ligase by these derivatives, three types of protein substrates could be distinguished. Type I substrates are proteins that have a basic NH2-terminal residue (such as ribonuclease and lysozyme); these are specifically inhibited by derivatives of the 3 basic amino acids (His, Arg, and Lys) with respect to degradation, ligation to ubiquitin, and binding to ligase. Type II substrates (such as beta-lactoglobulin or pepsinogen, that have a Leu residue at the NH2 terminus) are not affected by the above compounds, but are specifically inhibited by derivatives of bulky hydrophobic amino acids (Leu, Trp, Phe, and Tyr). In these cases, the amino acid derivatives apparently act as specific inhibitors of the binding of the NH2-terminal residue of proteins, as indicated by the following observations: (a) derivatives in which the alpha-NH2 group is blocked were inactive and (b) in dipeptides, the inhibitory amino acid residue had to be at the NH2-terminal position. An additional class (Type III) of substrates comprises proteins that have neither basic nor bulky hydrophobic NH2-terminal amino acid residues; the binding of these proteins is not inhibited by homologous amino acid derivatives that have NH2-terminal residues similar to that of the protein. It is concluded that Type I and Type II proteins bind to distinct and separate subsites of the ligase, specific for basic or bulky hydrophobic NH2-terminal residues, respectively. On the other hand, Type III proteins apparently predominantly interact with the ligase at regions of the protein molecule other than the NH2-terminal residue.     

Characterization of beta 1----4 galactosyltransferase purified from rat liver microsomes.

beta 1----4 Galactosyltransferase was purified from rat liver microsomes. Catalytic properties of the enzyme resembled those of previously purified soluble and membrane-bound beta 1----4 galactosyltransferases. The enzyme purified in the present study showed a major band around a molecular weight of 53,000 on SDS-PAGE. The NH2-terminal sequence of the enzyme was determined up to the 20th residue. The sequence was identical to the amino acid sequence from Ala-13 to Lys-32 deduced from mouse beta 1----4 galactosyltransferase cDNA. These results suggest that most of the mature enzyme in rat liver microsomes is produced by removal of the NH2-terminal 12 amino acids from a precursor polypeptide.     

Human adenine phosphoribosyltransferase. Complete amino acid sequence of the erythrocyte enzyme

We defined the amino acid sequence of adenine phosphoribosyltransferase isolated from human erythrocytes. Peptide fragments formed by cleavage at arginine, lysine, glutamic acid, and methionine were purified by high pressure liquid chromatography and sequenced by manual Edman degradation. The complete primary structure of human adenine phosphoribosyltransferase was established by sequence analysis of 19 peptide fragments. Presumed homology between the human and rodent enzymes was used to order fragments that had inadequate overlapping sequences. The enzyme has 179 residues with a calculated subunit molecular weight of 19,481. Mass spectrometry indicated that the NH2-terminal residue is acetylated. Human adenine phosphoribosyltransferase has sequence homology with xanthine-guanine phosphoribosyltransferase from Escherichia coli in 110-amino acid region encompassing the NH2-terminal section of the enzyme.     

Beta-ketoadipate enol-lactone hydrolases I and II from Acinetobacter calcoaceticus.

Beta-Ketoadipate enol-lactone hydrolase catalyzes a common step in the utilization of protocatechuate and cis,cis-muconate by bacteria. Either of the two compounds elicits the synthesize of an enol-lactone hydrolase in Acinetobacter. The enol-lactone hydrolase that is induced by each compound was purified, and the properties of the proteins were compared. Both enzymes appear to be dimers with molecular weights of approximately 25,000. The amino acid compositions of the enzymes differ, and the two proteins do not cross-react serologically. The NH2-terminal amino acid residue of the protocatechuate-induced enol-lactone hydrolase (ELH I) is methionine and the NH2-terminal amino acid residue of the cis,cis-muconate-induced enol-lactone hydrolase (ELH II) is proline. Therefore, ELH I and ELH II appear to be the products of different structural genes. The serological specificity of ELH I and ELH II made it possible to demonstrate the mutually independent regulation of their synthesis in wild type cells and in constitutive mutant strains. The synthesis of ELH I is not impaired in mutant strains that cannot synthesize ELH II. The rapid characterization of mutant strains that produce ELH I or ELH II constitutively was made possible by the development of pH indicator enzyme assays that were performed with toluenized cells. cis,trans-Muconate, which does not support the growth of Acinetobacter, elicits the synthesis of the enzymes that normally are induced by cis,cis-muconate to 20% of fully induced levels.     

Localization of the alpha-chain cross-link acceptor sites of human fibrin

The potential cross-link acceptor sites of fibrin were specifically labeled with the fluorescent, substitute cross-link donor monodansyl cadaverine (MDC). Several fluorescent alpha-chain peptides generated from enzymatic and cyanogen bromide (CNBr) cleavage of the labeled fibrin were identified by sodium dodecyl sulfate disc gel electrophoresis; they were isolated and then characterized by amino acid analysis, NH2-terminal sequence analysis, and chromatographic and electrophoretic analyses of their digestion products. Ancrod cleavage of MDC-labeled fibrin produced a series of six alpha-chain peptides of molecular weights 34,000 to 12,000, each of which contained an MDC-labeled acceptor site, and an NH2-terminal alpha-chain derivative of molecular weight 37,500. The latter remains disulfide bound in the residual fibrin and has two MDC-labeled sit-s which are separable by CNBr cleavage. Mild plasmin digestion of MDC-labeled fibrin generated fluorescent alpha-chain peptides of molecular weights 45,000, 42,000, 35,000, 23,000, 21,000, and 2,500 in the supernatant and a nonfluorescent NH2-terminal alpha-chain derivative of molecular weight 25,000 which remained in the insoluble residual fibrin. The alignment of these plasmic supernatant peptides was determined from NH2-terminal sequence analyses which indicated that an MDC acceptor site was located at approximately residue 255 of the Aalpha-chain. Cleavage of the MDC-labeled alpha-chain by CNBr, however, localized most of its fluorescence (approximately 80%) to a fragment of molecular weight 29,000 which had the same NH2-terminal sequence as the labeled plasmic peptide of molecular weight 21,000. Both peptides were cleaved by ancrod into two acceptor site-containing peptides of approximately equal fluorescence. The preliminary NH2-terminal sequence analyses of these peptides, when combined with the above findings, indicated that these two other cross-link acceptor sites are in a peptide segment which comprises the middle 17% of the Aalpha-chain.     

Two genes encoding fruit body lectins of Pleurotus cornucopiae: sequence similarity with the lectin of a nematode-trapping fungus

Our previous studies on the fruit body lectin of Pleurotus cornucopiae revealed the existence of three isolectins, composed of two homodimers and one heterodimer of 16- and 15-kDa subunits. In this study, two genes encoding the lectins were cloned and characterized. Both genes encoded 144 amino acids and only 5 amino acids were different within the coding region, but the nucleotide sequences of the 5'-upstream and 3'-downstream regions differed extensively. Southern hybridization with gene-specific probes showed that one gene encoded the 16-kDa and the other encoded the 15-kDa subunit. Functional lectins were synthesized in Escherichia coli under the direction of these genes. On SDS-PAGE, the recombinant lectins showed the same banding patterns as the native lectins. In amino acid sequence, these lectins showed extensive similarity with the lectin from a nematode-trapping ascomycete fungus, Arthrobotrys oligospora, suggesting that the lectins might also function in capturing nematodes.