Crystal structure of α-galactosidase from Lactobacillus acidophilus NCFM: insight into tetramer formation and substrate binding

Lactobacillus acidophilus NCFM is a probiotic bacterium known for its beneficial effects on human health. The importance of α-galactosidases (α-Gals) for growth of probiotic organisms on oligosaccharides of the raffinose family present in many foods is increasingly recognized. Here, the crystal structure of α-Gal from L. acidophilus NCFM (LaMel36A) of glycoside hydrolase (GH) family 36 (GH36) is determined by single-wavelength anomalous dispersion. In addition, a 1.58-Å-resolution crystallographic complex with α-d-galactose at substrate binding subsite − 1 was determined. LaMel36A has a large N-terminal twisted β-sandwich domain, connected by a long α-helix to the catalytic (β/α)₈-barrel domain, and a C-terminal β-sheet domain. Four identical monomers form a tightly packed tetramer where three monomers contribute to the structural integrity of the active site in each monomer. Structural comparison of LaMel36A with the monomeric Thermotoga maritima α-Gal (TmGal36A) reveals that O2 of α-d-galactose in LaMel36A interacts with a backbone nitrogen in a glycine-rich loop of the catalytic domain, whereas the corresponding atom in TmGal36A is from a tryptophan side chain belonging to the N-terminal domain. Thus, two distinctly different structural motifs participate in substrate recognition. The tetrameric LaMel36A furthermore has a much deeper active site than the monomeric TmGal36A, which possibly modulates substrate specificity. Sequence analysis of GH36, inspired by the observed structural differences, results in four distinct subgroups having clearly different active-site sequence motifs. This novel subdivision incorporates functional and architectural features and may aid further biochemical and structural analyses within GH36.     

DNA-binding specificity of the Lon protease α-domain from Brevibacillus thermoruber WR-249

Lon protease has been well studied in many aspects; however, the DNA-binding specificity of Lon in prokaryotes has not been clearly identified. Here we examined the DNA-binding activity of Lon protease α-domains from Brevibacillus thermoruber (Bt), Bacillus subtilis (Bs), and Escherichia coli (Ec). MALDI-TOF mass spectroscopy showed that the α-domain from Bt-Lon binds to the duplex nucleotide sequence 5′-CTGTTAGCGGGC-3′ (ms1) and protected it from DNase I digestion. Surface plasmon resonance showed that the Bt-Lon α-domain binds with ms1 double-stranded DNA tighter than Bs- and Ec-Lon α-domains, whereas the Bt-Lon α-domain has dramatically lower affinity for double-stranded DNA with 0 and 50% identity to the ms1 binding sequence. Our results indicated that Bt-Lon α-domain plays a critical role with ms1 sequence in the DNA-binding specificity.     

Crystal Structure of the GalNAc/Gal-Specific Agglutinin from the Phytopathogenic Ascomycete Sclerotinia sclerotiorum Reveals Novel Adaptation of a β-Trefoil Domain

A lectin from the phytopathogenic ascomycete Sclerotinia sclerotiorum that shares only weak sequence similarity with characterized fungal lectins has recently been identified. S. sclerotiorum agglutinin (SSA) is a homodimeric protein consisting of two identical subunits of ∼ 17 kDa and displays specificity primarily towards Gal/GalNAc. Glycan array screening indicates that SSA readily interacts with Gal/GalNAc-bearing glycan chains. The crystal structures of SSA in the ligand-free form and in complex with the Gal-β1,3-GalNAc (T-antigen) disaccharide have been determined at 1.6 and 1.97 Å resolution, respectively. SSA adopts a β-trefoil domain as previously identified for other carbohydrate-binding proteins of the ricin B-like lectin superfamily and accommodates terminal non-reducing galactosyl and N-acetylgalactosaminyl glycans. Unlike other structurally related lectins, SSA contains a single carbohydrate-binding site at site α. SSA reveals a novel dimeric assembly markedly dissimilar to those described earlier for ricin-type lectins. The present structure exemplifies the adaptability of the β-trefoil domain in the evolution of fungal lectins.     

Purification and Characterization of a Novel β-<Emphasis Type="SmallCaps">d</Emphasis>-Galactosides-Specific Lectin from <Emphasis Type="Italic">Clitoria ternatea</Emphasis>

A lectin present in seeds of Clitoria ternatea agglutinated trypsin-treated human B erythrocytes. The sugar specificity assay indicated that lectin belongs to Gal/Gal NAc-specific group. Hence the lectin, designated C. ternatea agglutinin (CTA), was purified by the combination of acetic acid precipitation, salt fractionation and affinity chromatography. HPLC gel filtration, SDS-polyacrylamide gel electrophoresis and mass spectrometry indicated that the native lectin is composed of two identical subunits of molecular weight 34.7 kDa associated by non covalent bonds. The N-terminal sequence of CTA shared homology with Glycine max and Pisum sativum. Complete sequence was also found to be homologous to S-64 protein of Glycine max, suggesting that CTA probably exhibits both hemagglutination and probably sugar uptake activity. The carbohydrate binding specificity of the lectin was investigated by quantitative turbidity measurements, and percent inhibition assays. Based on these assays, we conclude that CTA binds β-d-galactosides, and also may has an extended specificity towards non-reducing terminal Neu5Acα2,6Gal.     

Purification,characterization and cloning of a ricin B-like lectin from mushroom Clitocybe nebularis with antiproliferative activity against human leukemic T cells

Background

Lectins are a diverse group of carbohydrate-binding proteins exhibiting numerous biological activities and functions.

Methods

Two-step serial carbohydrate affinity chromatography was used to isolate a lectin from the edible mushroom clouded agaric (Clitocybe nebularis). It was characterized biochemically, its gene and cDNA cloned and the deduced amino acid sequence analyzed. Its activity was tested by hemagglutination assay and carbohydrate-binding specificity determined by glycan microarray analysis. Its effect on proliferation of several human cell lines was determined by MTS assay.

Results

A homodimeric lectin with 15.9-kDa subunits agglutinates human group A, followed by B, O, and bovine erythrocytes. Hemagglutination was inhibited by glycoprotein asialofetuin and lactose. Glycan microarray analysis revealed that the lectin recognizes human blood group A determinant GalNAcα1–3(Fucα1–2)Galβ-containing carbohydrates, and GalNAcβ1–4GlcNAc (N,N'-diacetyllactosediamine). The lectin exerts antiproliferative activity specific to human leukemic T cells.

Conclusions

The protein belongs to the ricin B-like lectin superfamily, and has been designated as C. nebularis lectin (CNL). Its antiproliferative effect appears to be elicited by binding to carbohydrate receptors on human leukemic T cells.

General significance

CNL is one of the few mushroom ricin B-like lectins that have been identified and the only one so far shown to possess immunomodulatory properties.     

Structural insights into substrate specificity of crotonase from the n-butanol producing bacterium Clostridium acetobutylicum

Crotonase from Clostridium acetobutylicum (CaCRT) is an enzyme that catalyzes the dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA in the n-butanol biosynthetic pathway. To investigate the molecular mechanism underlying n-butanol biosynthesis, we determined the crystal structures of the CaCRT protein in apo- and acetoacetyl-CoA bound forms. Similar to other canonical crotonase enzymes, CaCRT forms a hexamer by the dimerization of two trimers. A crystal structure of CaCRT in complex with acetoacetyl-CoA revealed that Ser69 and Ala24 to be signature residues of CaCRT, which results in a distinct ADP binding mode wherein the ADP moiety is bound at a different position compared with other crotonases. We also revealed that the substrate specificity of crotonase enzymes is determined by both the structural feature of the α3 helix region and the residues contributing the enoyl-CoA binding pocket. A tight formed α3 helix and two phenylalanine residues, Phe143 and Phe233, aid CaCRT to accommodate crotonyl-CoA as the substrate. The key residues involved in substrate binding, enzyme catalysis and substrate specificity were confirmed by site-directed mutagenesis.     

Crystal Structure of an Essential Enzyme in Seed Starch Degradation: Barley Limit Dextrinase in Complex with Cyclodextrins

Barley limit dextrinase [Hordeum vulgare limit dextrinase (HvLD)] catalyzes the hydrolysis of α-1,6 glucosidic linkages in limit dextrins. This activity plays a role in starch degradation during germination and presumably in starch biosynthesis during grain filling. The crystal structures of HvLD in complex with the competitive inhibitors α-cyclodextrin (CD) and β-CD are solved and refined to 2.5 Å and 2.1 Å, respectively, and are the first structures of a limit dextrinase. HvLD belongs to glycoside hydrolase 13 family and is composed of four domains: an immunoglobulin-like N-terminal eight-stranded β-sandwich domain, a six-stranded β-sandwich domain belonging to the carbohydrate binding module 48 family, a catalytic (β/α)₈-like barrel domain that lacks α-helix 5, and a C-terminal eight-stranded β-sandwich domain of unknown function. The CDs are bound at the active site occupying carbohydrate binding subsites + 1 and + 2. A glycerol and three water molecules mimic a glucose residue at subsite − 1, thereby identifying residues involved in catalysis. The bulky Met440, a unique residue at its position among α-1,6 acting enzymes, obstructs subsite − 4. The steric hindrance observed is proposed to affect substrate specificity and to cause a low activity of HvLD towards amylopectin. An extended loop (Asp513-Asn520) between β5 and β6 of the catalytic domain also seems to influence substrate specificity and to give HvLD a higher affinity for α-CD than pullulanases. The crystal structures additionally provide new insight into cation sites and the concerted action of the battery of hydrolytic enzymes in starch degradation.     

Structural implications of Siglec-5-mediated sialoglycan recognition

Sialic acid (Sia) Ig-like binding lectins are important mediators of recognition and signaling events among myeloid cells. To investigate the molecular mechanism underlying sialic acid Ig-like lectin (Siglec) functions, we determined the crystal structure of the two N-terminal extracellular domains of human myeloid cell inhibitory receptor Siglec-5 (CD170) and its complexes with two sialylated carbohydrates. The native structure revealed an unusual conformation of the CC′ ligand specificity loop and a unique interdomain disulfide bond. The α(2,3)- and α(2,6)-sialyllactose complexed structures showed a conserved Sia recognition motif that involves both Arg124 and a portion of the G-strand in the V-set domain forming β-sheet-like hydrogen bonds with the glycerol side chain of the Sia. Only few protein contacts to the subterminal sugars are observed and mediated by the highly variable GG′ linker and CC′ loop. These structural observations, in conjunction with surface plasmon resonance binding assays, provide mechanistic insights into linkage-dependent Siglec carbohydrate recognition and suggest that Siglec-5 and other CD33-related Siglec receptors are more promiscuous in sialoglycan recognition than previously understood.     

Ginger rhizome as a potential source of milk coagulating cysteine protease

A milk coagulating protease was purified ∼10.2-fold to apparent homogeneity from ginger rhizomes in 34.9% recovery using ammonium sulfate fractionation, together with ion exchange and size exclusion chromatographic techniques. The molecular mass of the purified protease was estimated to be ∼36 kDa by SDS-PAGE, and exhibited a pI of 4.3. It is a glycoprotein with 3% carbohydrate content. The purified enzyme showed maximum activity at pH 5.5 and at a temperature of ∼60 °C. Its protease activity was strongly inhibited by iodoacetamide, E-64, PCMB, Hg²⁺ and Cu²⁺. Inhibition studies and N-terminal sequence classified the enzyme as a member of the cysteine proteases. The cleavage capability of the isolated enzyme was higher for α_s-casein followed by β- and κ-casein. The purified enzyme differed in molecular mass, pI, carbohydrate content, and N-terminal sequence from previously reported ginger proteases. These results indicate that the purified protease may have potential application as a rennet substitute in the dairy industry.     

Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii

An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60 °C and 4.0, respectively. The enzyme was stable for 1 h at 55 °C, showing a t₅₀ of 53 min at 60 °C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K_m of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase).     

Characterization of a recombinant α-glucuronidase from Aspergillus fumigatus

The degradation of xylan requires the action of glycanases and esterases which hydrolyse, in a synergistic fashion, the main chain and the different substituents which decorate its structure. Among the xylanolytic enzymes acting on side-chains are the α-glucuronidases (AguA) (E.C. 3.2.1.139) which release methyl glucuronic acid residues. These are the least studies among the xylanolytic enzymes. In this work, the gene and cDNA of an α-glucuronidase from a newly isolated strain of Aspergillus fumigatus have been sequenced, and the gene has been expressed in Pichia pastoris. The gene is 2523 bp long, has no introns and codes for a protein of 840 amino acid residues including a putative signal peptide of 19 residues. The mature protein has a calculated molecular weight of 91 725 and shows 99 % identity with a putative α-glucuronidase from A. fumigatus A1163. The recombinant enzyme was expressed with a histidine tag and was purified to near homogeneity with a nickel nitriloacetic acid (Ni-NTA) column. The purified enzyme has a molecular weight near 100 000. It is inactive using birchwood glucuronoxylan as substrate. Activity is observed in the presence of xylooligosaccharides generated from this substrate by a family 10 endoxylanase and when a mixture of aldouronic acids are used as substrates. If, instead, family 11 endoxylanase is used to generate oligosaccharides, no activity is detected, indicating a different specificity in the cleavage of xylan by family 10 and 11 endoxylanases. Enzyme activity is optimal at 37 °C and pH 4.5–5. The enzyme binds cellulose, thus it likely possesses a carbohydrate binding module. Based on its properties and sequence similarities the catalytic module of the newly described α-glucuronidase can be classified in family 67 of the glycosyl hydrolases. The recombinant enzyme may be useful for biotechnological applications of α-glucuronidases.     

Recognition intensities of submolecular structures,mammalian glyco-structural units,ligand cluster and polyvalency in abrin-a-carbohydrate interactions

Abrin-a is the most toxic fraction of lectins isolated from Abrus precatorius seeds and belongs to the family of type 2 ribosome inactivating proteins (RIP). This toxin may act as a defense molecule in plants against viruses, fungi and insects, where attachment of abrin-a to the exposed glycans on the surface of target cells is the crucial and initial step of its cytotoxicity. Although it has been studied for over four decades, the recognition factors involved in abrin-a-carbohydrate interaction remains to be clarified. In this study, roles of mammalian glyco-structural units, ligand clusters and polyvalency in abrin-a recognition were comprehensively analyzed by enzyme-linked lectinosorbent binding and inhibition assays. The results indicate that: (i) this toxin prefers oligosaccharides having α-anomer of galactose (Gal) at the non-reducing terminal than the corresponding β-anomer; (ii) Galα1-3Galα1- (B_α), Galα1-4Gal (E), Galβ1-3GalNAc (T) and Galβ1-3/4GlcNAc (I/II) related oligosaccharides were the active glyco-structural units; (iii) tri-antennary II_β, prepared from N-glycan of asialo fetuin, played a dominant role in recognition; (iv) many high-density polyvalent I_β/II_β and E_β glycotopes enhanced the reactivity; (v) the carbohydrate recognition domain of abrin-a is proposed to be a combination of a small cavity type of Gal as major site and a groove type of additional one to tetrasaccharides as subsites with a preference of α1-3/4/6Gal, β1-3GalNAc, β1-3/4/6GlcNAc, β1-4/6Glc, β1-3DAra and β1-4Man as subterminal sugars; (vi) size of the carbohydrate recognition domain may be as large enough to accommodate a linear pentasaccharide and complementary to Galα1-3Galβ1-4GlcNAc β1-3Galβ1-4Glc (gailipenta) sequence. A comparison of the recognition factors and combining sites of abrin-a with ricin, another highly toxic lectin, was also performed to further understand the differences in recognition factors between these two type 2 RIPs.     

Effects of Lectins on initial attachment of cariogenic <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

Oral bacteria initiate biofilm formation by attaching to tooth surfaces via an interaction of a lectin-like bacterial protein with carbohydrate chains on the pellicle. This study aimed to find naturally derived lectins that inhibit the initial attachment of a cariogenic bacterial species, Streptococcus mutans (S. mutans), to carbohydrate chains in saliva in vitro. Seventy kinds of lectins were screened for candidate motifs that inhibit the attachment of S. mutans ATCC 25175 to a saliva-coated culture plate. The inhibitory effect of the lectins on attachment of the S. mutans to the plates was quantified by crystal violet staining, and the biofilm was observed under a scanning electron microscope (SEM). Surface plasmon resonance (SPR) analysis was performed to examine the binding of S. mutans to carbohydrate chains and the binding of candidate lectins to carbohydrate chains, respectively. Moreover, binding assay between the biotinylated-lectins and the saliva components was conducted to measure the lectin binding. Lectins recognizing a salivary carbohydrate chain, Galβ1-3GalNAc, inhibited the binding of S. mutans to the plate. In particular, Agaricus bisporus agglutinin (ABA) markedly inhibited the binding. This inhibition was confirmed by SEM observation. SPR analysis indicated that S. mutans strongly binds to Galβ1-3GalNAc, and ABA binds to Galβ1-3GalNAc. Finally, the biotinylated Galβ1-3GalNAc-binding lectins including ABA demonstrated marked binding to the saliva components. These results suggest that ABA lectin inhibited the attachment of S. mutans to Galβ1-3GalNAc in saliva and ABA can be useful as a potent inhibitor for initial attachment of oral bacteria and biofilm formation.     

NMR investigations of protein–carbohydrate interactions: insights into the topology of the bound conformation of a lactose isomer and β-galactosyl xyloses to mistletoe lectin and galectin-1

A hallmark of oligosaccharides is their often limited spatial flexibility, allowing them to access a distinct set of conformers in solution. Viewing each individual or even the complete ensemble of conformations as potential binding partner(s) for lectins in protein–carbohydrate interactions, it is pertinent to address the question on the characteristics of bound state conformation(s) in solution. Also, it is possible that entering the lectin’s binding site distorts the low-energy topology of a glycosidic linkage. As a step to delineate the strategy of ligand selection for galactosides, a common physiological docking point, we have performed a NMR study on two non-homologous lectins showing identical monosaccharide specificity. Thus, the conformation of lactose analogues bound to bovine heart galectin-1 and to mistletoe lectin in solution has been determined by transferred nuclear Overhauser effect measurements. It is demonstrated that the lectins select the syn conformation of lactose and various structural analogues (Galβ(1→4)Xyl, Galβ(1→3)Xyl, Galβ(1→2)Xyl, and Galβ(1→3)Glc) from the ensemble of presented conformations. No evidence for conformational distortion was obtained. Docking of the analogues to the modeled binding sites furnishes explanations, in structural terms, for exclusive recognition of the syn conformer despite the non-homologous design of the binding sites.     

Isolation, structural characterization and antioxidant activity of a new water-soluble polysaccharide from Acanthophyllum bracteatum roots

ABPS-1, a new water-soluble polysaccharide with molecular weight of 26 kDa and a specific optical rotation of +170° (c 1.0, H₂O), was extracted from the roots of Acanthophyllum bracteatum by warm water and further successively purified through DEAE-cellulose A52 and Sephadex G-100 columns. Monosaccharide analysis revealed that the ABPS-1 was composed of Glc, Gal and Ara with a relative molar ratio of 1.4:5.2:1.0. Its structural features were elucidated by a combination of FT-IR, methylation and GC-MS analysis, periodate oxidation and Smith degradation, partial acid hydrolysis and ¹³C and ¹H NMR spectroscopy. The data obtained indicate that ABPS-1 possessed a backbone of α-(1 → 6)-linked Gal with branches attached to O-2 by α-1 → linked Glc and at O-3 by α-1 → linked Gal and by α-(1 → 3)-linked Ara. The in vitro antioxidant activity showed that ABPS-1 possesses DPPH radical-scavenging activity in a concentration-dependent manner with an EC₅₀ value of 2.6 mg/ml.     

A mushroom lectin from ascomycete Cordyceps militaris

A mushroom lectin has been purified from ascomycete Cordyceps militaris, which is one of the most popular mushrooms in eastern Asia used as a nutraceutical and in traditional Chinese medicine. This lectin, designated CML, exhibited hemagglutination activity in mouse and rat erythrocytes, but not in human ABO erythrocytes. SDS-PAGE of CML revealed a single band with a molecular mass of 31.0 kDa under both nonreducing and reducing conditions that was stained by silver nitrate, and a 31.4 kDa peak in a Superdex-200 HR gel-filtration column. The hemagglutination activity was inhibited by sialoglycoproteins, but not in by mono- or disaccharides, asialoglycoproteins, or de-O-acetylated glycoprotein. The activity was maximal at pH 6.0–9.1 and at temperatures below 50 °C. Circular dichroism spectrum analysis revealed that CML comprises 27% α-helix, 12% β-sheets, 29% β-turns, and 32% random coils. Its binding specificity and secondary structure are similar to those of a fungal lectin from Arthrobotrys oligospora. However, the N-terminal amino acid sequence of CML differs greatly from those of other lectins. CML exhibits mitogenic activity against mouse splenocytes.     

Toxin A from Clostridium difficile binds to rabbit erythrocyte glycolipids with terminal Gal alpha 1-3Gal beta 1-4GlcNAc sequences

The binding of Toxin A isolated from Clostridium difficile to rabbit erythrocyte glycolipids has been studied. Total lipid extracts from rabbit erythrocytes were subjected to thin-layer chromatography and toxin-binding glycolipids detected by using 125I-labeled Toxin A in a direct binding overlay technique. Two major and several minor toxin-binding glycolipids were detected in rabbit erythrocytes by this method. The results of structural analyses of the major toxin-binding glycolipids were consistent with a pentasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) and a branched decasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3[Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) previously identified as the two most abundant glycolipids in rabbit erythrocytes. 125I-Toxin A binding to these glycolipids could be inhibited by bovine thyroglobulin, monospecific antiserum to the toxin, or by treatment of the glycolipids with alpha-galactosidase. The absence of toxin interaction with isoglobotriaosylceramide (Gal alpha 1-3Gal beta 1-4Glc-Cer) isolated from canine intestine suggested that the GlcNAc residue present in the terminal Gal alpha 1-3Gal beta 1-4GLcNAc sequence common to all known toxin binding glycoconjugates is required for carbohydrate-specific recognition by Toxin A. These observations are consistent with the proposed carbohydrate binding specificity of Toxin A for the nonreducing terminal sequence, Gal alpha 1-3Gal beta 1-4GlcNAc.     

Chemical structures of oligosaccharides in milk of the raccoon (<Emphasis Type="Italic">Procyon lotor</Emphasis>)

In this study on milk saccharides of the raccoon (Procyonidae: Carnivora), free lactose was found to be a minor constituent among a variety of neutral and acidic oligosaccharides, which predominated over lactose. The milk oligosaccharides were isolated from the carbohydrate fractions of each of four samples of raccoon milk and their chemical structures determined by ¹H-NMR and MALDI-TOF mass spectroscopies. The structures of the four neutral milk oligosaccharides were Fuc(α1–2)Gal(β1–4)Glc (2′-fucosyllactose), Fuc(α1–2)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc (lacto-N-fucopentaose IV), Fuc(α1–2)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc (fucosyl para lacto-N-neohexaose) and Fuc(α1–2)Gal(β1–4)GlcNAc(β1–3)[Fuc(α1–2)Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (difucosyl lacto-N-neohexaose). No type I oligosaccharides, which contain Gal(β1–3)GlcNAc units, were detected, but type 2 saccharides, which contain Gal(β1–4)GlcNAc units were present. The monosaccharide compositions of two of the acidic oligosaccharides were [Neu5Ac]₁[Hex]₆[HexNAc]₄[deoxy Hex]₂, while those of another two were [Neu5Ac]₁[Hex]₈[HexNAc]₆[deoxy Hex]_3. These acidic oligosaccharides contained α(2–3) or α(2–6) linked Neu5Ac, non reducing α(1–2) linked Fuc, poly N-acetyllactosamine (Gal(β1–4)GlcNAc) and reducing lactose.     

Characterization of α-mannosidase from Erythrina indica seeds and influence of endogenous lectin on its activity

α-mannosidase from Erythrina indica seeds is a Zn²⁺ dependent glycoprotein with 8.6% carbohydrate. The enzyme has a temperature optimum of 50 °C and energy of activation calculated from Arrhenius plot was found to be 23 kJ mol^− 1. N-terminal sequence up to five amino acid residues was found to be DTQEN (Asp, Thr, Gln, Glu, and Asn). In chemical modification studies treatment of the enzyme with NBS led to total loss of enzyme activity and modification of a single tryptophan residue led to inactivation. Fluorescence studies over a pH range of 3–8 have shown tryptophan residue to be in highly hydrophobic environment and pH change did not bring about any appreciable change in its environment. Far-UV CD spectrum indicated predominance of α-helical structure in the enzyme. α-Mannosidase from E indica exhibits immunological identity with α-mannosidase from Canavalia ensiformis but not with the same enzyme from Glycine max and Cicer arietinum. Incubation of E. indica seed lectin with α-mannosidase resulted in 35% increase in its activity, while no such activation was observed for acid phosphatase from E. indica. Lectin induced activation of α-mannosidase could be completely abolished in presence of lactose, a sugar specific for lectin.