Distribution of the glycoconjugate oligosaccharides in the human placenta from pregnancies complicated by altered glycemia: lectin histochemistry

The aim of this study was to investigate the distribution of the oligosaccharides of the glycoconjugates in placentas from pregnancies complicated by different degree of altered glycaemia. Placentas from women with physiological pregnancies (group 1), with pregnancies complicated by minor degree of glucose intolerance (group 2) and with pregnancies complicated by gestational diabetes mellitus (GDM) treated with insulin (group 3) were collected. Ten lectins were used (ConA, WGA, PNA, SBA, DBA, LTA, UEA I, GSL II, MAL II and SNA) in combination with chemical and enzymatic treatments. The data showed a decrease of sialic acid linked α(2–6) to galactose/N-acetyl-d-galactosamine and an increase of N-acetyl-d-glucosamine in the placentas of the pathological groups, in particular the group 3, comparing to the group 1. A decrease of l-fucose (LTA) and d-galactose-(β1–3)-N-acetyl-d-galactosamine, and an increase and/or appearance of l-fucose (UEA I) and N-acetyl-d-galactosamine were observed in both the pathological groups, particularly in the group 2, with respect to the group 1. In GDM, and even in pregnancies with a simple alteration of maternal glycaemia, the changes in the distribution of oligosaccharides could be related to alteration of the structure and functionality of the placenta.     

The cytochemistry of glycoconjugates in the planum nasolabial gland of the goat as studied by electron microscopic methods

Summary In the planum nasolabial glands of the goat, glycoconjugates of glandular and duct cells have been studied by means of a series of electron microscopic cytochemical methods. In the glandular cells glycoconjugates with vicinal diol groupings were present in secretory granules, certain elements of the Golgi complex, lysosome-like dense bodies, the surface coat of the plasma membrane, the majority of intracellular cytomembranes, glycogen particles and the basal lamina. In duct cells, glycoconjugates with the same properties were localized in similar ultrastructures, except for secretory granules, which were not detected in these cells. By lectin cytochemistry, glycoconjugates in glandular cell secretory granules contained a variety of saccharide residues such as -d-mannose, -d-glucose,N-acetyl-d-glucosamine and -l-fucose. The cytochemical properties of the secretory glycoconjugates are discussed in relation to the physiological functions performed by the planum nasolabial glands in the goat.     

Ultrastructural changes in the oviductal epithelium of Merino ewes during the estrous cycle

Isthmic and ampullary oviductal epithelia sampled from Merino ewes at days -1, 1, 3, and 10 of the estrous cycle (estrus = day 0) were studied by scanning and transmission electron microscopy after fixation by vascular perfusion. Secretory cells, ciliated cells, and lymphocytelike basal cells were observed in both isthmic and ampullary epithelium at all stages of the estrous cycle studied and their ultrastructural features were analyzed. Synthesis of lamellated secretory granules occurred in the ampullary secretory cells during the follicular and early luteal phases, and their contents were released by exocytosis into the oviductal lumen during the luteal phase. Granule release was associated with nucleated apical protrusion of these cells into the oviductal lumen. No such secretory activity was displayed by isthmic secretory cells even though a few cells contained nonlamellated granules. Apocrine release of apical vesicles and accompanying cytoplasmic material from apical protrusions of ciliated cells occurred in the isthmus around estrus but not in the ampulla. This unexpected feature has not previously been reported in any other mammal. Dendritic basal cells were distinguished in the lower part of the epithelium by their heterochromatic nuclei, electron-lucent cytoplasm, and lack of attachment zones. No migration of basal cells was observed, and their ultrastructural features were similar in the ampulla and isthmus and at all stages of the estrous cycle examined. The function of these lymphocytelike cells in the epithelium is uncertain, but the presence of phagocytic bodies and lysosomes in 20% of them may indicate a phagocytic role.     

Sperm binding to oviductal epithelial cells in the rat: role of sialic acid residues on the epithelial surface and sialic acid-binding sites on the sperm surface

The aim of this study was to assess the participation of carbohydrate residues in the adhesion of spermatozoa to the oviductal epithelium in the rat. We first examined, by lectin labeling, the distribution of glycoconjugates in rat oviducts obtained under various hormonal environments. Several classes of glycoconjugates were abundant in the epithelium, and the expression of some of these molecules varied differentially in ampulla and isthmus, along the estrous cycle and with estradiol and progesterone treatment. Proestrous rats were intraoviductally injected with lectins Dolichos biflorus, Erythrina cristagalli, Helix pomatia, Arachis hypogea, Ulex europaeus I, Triticum vulgaris, or Tritrichomonas mobilensis and were inseminated with 10-20 million epididymal spermatozoa in each uterine horn. Three hours later, the total number of spermatozoa present in the oviduct and the proportion adhering to the epithelium were determined. Intraoviductal administration of lectins did not affect the total number of spermatozoa recovered from the oviduct and only the sialic acid-binding lectin TML decreased the percentage of sperm cells adhering to the epithelium. The involvement of sialic acid in sperm-oviduct adhesion was further explored, inseminating spermatozoa preincubated with mannose, galactose, sialic acid, fucose, fetuin, or asialofetuin. Sialic acid and fetuin inhibited sperm-oviduct binding while other carbohydrates had no effect. Using TML lectin immunohistochemistry, we found that sialic acid-rich glycoconjugates are equally localized in the epithelium of ampulla and isthmus of proestrous rats. The electrophoretic pattern of sialic acid-rich glycoproteins of the epithelium showed 15 major protein bands, for which molecular mass ranged from 200 to 50 kDa with no difference between ampulla and isthmus or between estrous cycle stages. Binding sites for sialic acid-fluorescein isothiocyanate were demonstrated on the surface of rat spermatozoa, and biotinylated sialic acid recognized 11 plasma membrane proteins of sperm cells. These groups of sialic acid-rich glycoproteins in the oviductal epithelium and of sialic acid-binding proteins in the plasma membrane of sperm cells are good candidates for further studies to characterize the molecules responsible for sperm binding. We conclude that there are segment-specific changes of sugar residues present in the oviductal epithelium associated with different endocrine environments. Sperm-oviduct adhesion in the rat occurs by interaction of sialoglycoconjugates present in the epithelial cells with sialic acid-binding proteins on the sperm surface. This replicates the situation previously found in hamsters, disclosing for the first time that species-specificity in the sugar involved in sperm binding is not absolute.     

Lectinhistochemical demonstration of galactose- and N-acetyl-d-galactosamine residues in cells of the rat anterior pituitary

Summary Lectins are a useful tool for identification of differently glycosylated hypophyseal hormones, prohormones and glycoconjugates without hormone function. -d-galactose and -N-acetyl-d-galactosamine (GalNAc) containing glycoconjugates were identified by light microscopy with biotinylated lectins in immunocytochemically localized cells of the anterior pituitary of the rat. Galactose, histochemically detectable by the peanut lectin (PNA), was found at penultimate position of the carbohydrate chain after removal of sialic acid. Galactose containing cells correspond to gonadotrophs and thyrotrophs located mainly in medioanterior regions of the pituitary. The lectins from the soybean (SBA) and horse gram (DBA) both specific for GalNAc residues, are bound to round and also polygonal cells corresponding again to gonadotrophs and thyrotrophs.     

Ultracytochemistry of glycoproteins in the eccrine nasolabial glands of the North American raccoon (Procyon lotor)

The distribution and quality of glycoproteins was studied by means of electron microscopic cytochemical methods, particularly lectin cytochemistry, in the secretory cells of the eccrine nasolabial glands of the North American raccoon (Procyon lotor). In the dark and clear glandular cells, complex glycoconjugates were demonstrable, predominantly, in secretory granules, the cisternae of the Golgi apparatus, the surface coat of the plasma membrane, and as glycogen particles. Secretory granules found in the dark cells contained a variety of saccharide residues, such as α-d-mannose, β-d-galactose, β-N-acetyl-d-glucosamine and sialic acid. Several sugars were also detectable in the surface coat of the plasma membrane and the Golgi apparatus.The results obtained may be helpful to understand the specific functions of the glandular secretions of the raccoon nasolabial glands. These could be, particularly, binding of water on the snout surface and protection against microbial hazards, to maintain the structural and functional integrity of the relatively thin snout epidermis in carnivores.     

Asialo-α1-acid glycoprotein resialylated with 9-amino-5-N-acetyl-d-neuraminic acid is resistant towards bacterial,viral and mammalian sialidases

We demonstrate that 9-amino-NeuAc transferred to asialo-₁-acid glycoprotein resists cleavage by bacterial, viral and mammalian sialidases. This is the first synthetic sialic acid analogue, which can be activated and transferred to glycoprotein, but is not a sialidase (EC 3.2.1.18) substrate.Abbreviations HPLC high performance liquid chromatography - BSA bovine serum albumin - NeuAc N-acetyl-d-neuraminic acid, 5-acetamido-3,5-dideoxy-d-glycero-d-galacto-non-2-ulosonic acid - 9-Amino-NeuAc 9-amino-5-N-acetyl-d-neuraminic acid, 5-acetamido-9-trideoxy-d-glycero-d-galacto-non-2-ulosonic acid - CMP-NeuAc cytidine-5-monophospho-N-acetyl-d-neuraminic acid - CMP-9-amino-NeuAc cytidine-5-monophospho-9-amino-5-N-acetyl-d-neuraminic acid - 9-azido-NeuAc 5-acetamido-9-azido-3,5,9-trideoxy-d-glycero-d-galacto-non-2-ulosonic acid. Enzymes EC 3.2.1.18 sialidase, acylneuraminylhydrolase - EC 2.4.99.1 Galß1-4GlcNAc a(2-6)-sialytransferase     

Determination of the structure of sulfated tetra- and pentasaccharides obtained by alkaline borohydride degradation of hen ovomucin. A fast atom bombardment-mass spectrometric and1H-NMR spectroscopic study

Alkaline borohydride reductive cleavage of hen ovomucin resulted in the release of a series of neutral and acidic oligosaccharide-alditols.¹H-NMR spectroscopy in combination with fast ion bombardment-mass spectrometry in negative ion mode were used for investigation of the structures of three oligosaccharide-alditols. The following structures were established: Abbreviations NeuAc N-acetyl-d-neuraminic acid - Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Gal-NAc-ol N-acetyl-d-galactosaminitol - NMR nuclear magnetic resonance - FAB-MS fast atom bombardmentmass spectrometry     

Glycoconjugates in the secretory epithelium of the chicken mandibular gland

Summary In the secretory epithelium of the chicken mandibular gland, glycoconjugates have been studied by means of histochemical methods of light and electron microscopy. In light microscopy, a series of histochemical procedures have been employed which included lectin—peroxidase—diaminobenzidine methods and a digestion technique with neuraminidase or-amylase. In electron microscopy, a battery of methods were used that corresponded to those employed in light microscopy. In the secretory cells of the chicken mandibular gland, vicinal diol- and sulphate-containing glycoconjugates with sialic acid,-d-mannose,-d-glucose and-d-galactose residues were visualized and the possible histophysiological significances of such glycoconjugates were discussed with special reference to the functions of the salivary gland.     

Differential lectin binding patterns in the oviductal ampulla of the horse during oestrus

We investigated the oligosaccharide sequence of glycoconjugates, mainly sialoglycoconjugates, in the horse oviductal ampulla during oestrus by means of lectin and pre-lectin methods such as the KOH-neuraminidase procedure to remove sialic acid residues and incubation with N-glycosidase F to cleave N-linked glycans. Ciliated cells displayed N-linked oligosaccharides throughout the cytoplasm. The cilia glycocalyx expressed both N- and O-linked (mucin-type) oligosaccharides, both showing a high variety of terminal sequences. In the most non-ciliated cells, the whole cytoplasm contained N-linked oligosaccharides with terminal alphaGal as well as mucin-type glycans with terminal Forssman pentasaccharides. In a few scattered non-ciliated cells, the whole cytoplasm displayed sialylated N-linked oligosaccharides with terminal Neu5Ac-GalNAc and O-linked glycans terminating with neutral and/or alphaGalNAc, Neu5Ac alpha2,6Gal/GalNAc, Neu5AcGal beta1,3GalNAc. Supra-nuclear granules, probably Golgi zones, of non-ciliated cells showed mainly O-linked glycans rich in sialic acid residues. The luminal surface of non-ciliated cells showed N-linked oligosaccharides, containing terminal/internal alphaMan/alphaGlc, betaGlcNAc and terminal alphaGal, as well as mucin-type oligosaccharides terminating with a large variety of either neutral saccharides or sialylated sequences. Apical protrusions containing O-linked oligosaccharides with terminal Forssman pentasaccharide, Neu5Ac-Gal beta1,4GlcNAc, Neu5Ac-GalNAc were seen in non-ciliated cells scattered along the epithelium. These findings show the presence of sialoglycoconjugates in the oviductal ampulla epithelium of the mare and the existence of different lectin binding profiles between ciliated and non-ciliated (secretory) cells, as well as the presence of non-ciliated cell sub-types which might determine functional differences along the ampullary epithelium of mare oviduct.     

Histochemical study of Hurler's disease by the use of peroxidase-labelled lectins

Summary Peroxidase-labelled lectins specific for various carbohydrate residues were used as histochemical reagents in the investigation of Hurler's syndrome. Peanut lectin was used to detect terminald-galactose, wheatgerm lectin forN-acetyl-d-glucosamine, soybean lectin forN-acetyl-d-galactosamine,Tetragonolobus lotus lectin for -l-fucose andBandeiraea S. lectin for -d-galactose. It was found that Kupffer cells in the liver and splenic reticulo-endothelial cells contain acid mucopolysaccharides which bind lectins in paraffin sections after appropriate fixation. The pattern of lectin binding suggests that such cells contain significant amounts ofd-galactose,l-fucose,N-acetyl-d-galactosamine andN-acetyl-d-glucosamine. It is likely that the last named carbohydrate is present as a polymer. Neurones contain a different carbohydrate, rich in galactose and fucose but poor inN-acetyl-d-glucosamine. This compound is resistant to lipid extraction. Hepatocytes, as a rule, do not react with lectins, most likely because of loss of the more soluble mucopolysaccharides during fixation. The results are consistent with the biochemical data of Hurler's syndrome and indicate that lectins can be a useful tool for the investigation of the cytochemistry of storage disorders.     

Purification and properties of thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60

Thermostable N-acylamino acid recemase from Amycolatopsis sp. TS-1-60, a rare actinomycete strain selected for its ability to grow on agar plates incubated at 40° C, was purified to homogeneity and characterized. The relative molecular mass (M _r) of the native enzyme and the subunit was estimated to be 300 000 and 40 000 on gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis respectively. The isoelectric point (pI) of the enzyme was 4.2. The optimum temperature and pH were 50° C and 7.5 respectively. The enzyme was stable at 55° C for 30 min. The enzyme catalyzed the racemization of optically active N-acylamino acids such as N-acetyl-l-or d-methionine, N-acetyl-l-valine, N-acetyl-l-tyrosine and N-chloroacetyl-l-valine. In addition, the enzyme also catalyzed the recemization of the dipeptide l-alanyl-l-methionine. By contrast, the optically active amino acids, N-alkyl-amino acids and methyl and athyl ester derivatives of N-acetyl-d- and l-methionine were not racemized. The apparent K _m values for N-acetyl-l-methionine and N-acetyl-d-methionine were calculated to be 18.5 mM and 11.3 mM respectively. The enzyme activity was markedly enhanced by the addition of divalent metal ions such as Co²⁺, Mn²⁺ and Fe²⁺ and was inhibited by addition of EDTA and P-chloromercuribenzoic acid. The similarity between the NH₂-terminal amino acid sequence of the enzyme and that of Streptomyces atratus Y-53 [Tokuyama et al. (1994) Appl Microbiol Biotechnol 40:835–840] was above 80%.     

Camptosemin, a tetrameric lectin of Camptosema ellipticum: structural and functional analysis

Lectins have been classified into a structurally diverse group of proteins that bind carbohydrates and glycoconjugates with high specificity. They are extremely useful molecules in the characterization of saccharides, as drug delivery mediators, and even as cellular surface makers. In this study, we present camptosemin, a new lectin from Camptosema ellipticum. It was characterized as an N-acetyl-d-galactosamine-binding homo-tetrameric lectin, with a molecular weight around 26 kDa/monomers. The monomers were stable over a wide range of pH values and exhibited pH-dependent oligomerization. Camptosemin promoted adhesion of breast cancer cells and hemagglutination, and both activities were inhibited by its binding of sugar. The stability and unfolding/folding behavior of this lectin was characterized using fluorescence and far-UV circular dichroism spectroscopies. The results indicate that chemical unfolding of camptosemin proceeds as a two-state monomer-tetramer process. In addition, small-angle X-ray scattering shows that camptosemin behaves as a soluble and stable homo-tetramer molecule in solution.     

Detection and mapping of endogenous receptors for carrier-immobilized constituents of glycoconjugates (lectins) by labelled (neo)glycoproteins and by affinity chromatography in human adult mesencephalon,pons, medulla oblongata and cerebellum

Summary Different carrier-immobilized carbohydrate moieties were employed as tools to detect respective binding sites glycohistochemically and glycobiochemically. Besides ascertaining their presence the pattern of endogenous sugar receptors (lectins) in different regions of the human central nervous system was mapped to reveal any non-uniform expression. A strong and specific staining with biotinylated neoglycoproteins, exposing different sugar moieties as ligands, indicated the presence of sugar receptors in the nuclei, neuronal pathways and accessory structures such as ependyma cells, plexus chorioideus, intra- and extracerebral vessels and leptomeninges localized in the mesencephalon, in the pons, in the medulla oblongata and in the cerebellum. Significant differences were seen for various neuroanatomical regions like nerve cells in the basal and central regions of the nuclei pontis in the glycohistochemically detected level of expression of endogenous sugar receptors (lectins). The used approach with carbohydrate constituents of cellular glycoconjugates as ligands in search of specific receptors complemented studies on the localization of glycoconjugates with sugar-specific tools like plant lectins. Exemplary glycobiochemical investigations on the medulla oblongata and cerebellum were performed to investigate the molecular nature of sugar receptors detected glycohistochemically. Despite notable overall similarities, carbohydrate-binding proteins of differing molecular weight can be isolated from these two regions of the central nervous system, namely in the case of receptors with specificity to -galactoside termini, to N-acetyl-d-galactosamine and N-acetyl-d-glucosamine and to d-xylose. These combined glycohistochemical and glycobiochemical results serve as a guideline for exploring the physiological relevance of the detected regional differences.     

5-Bromo-indol-3-yl 5-acetamido-3,5-dideoxy-α-d-glycero-d-galactononulopyranosidonic acid,a novel chromogenic substrate for the staining of sialidase activity

The purification and characterisation of viral, bacterial and mammalian sialidases (EC 3.2.1.18, neuraminidases, neuraminosylglycohydrolases) prompted a search for a colorimetric technique to localize the enzymes on electropherograms. The 5-bromo substituted indol-3-yl -ketoside of 5-N-acetyl-d-neuraminic acid (BIN), the synthesis of which is described here, seemed to be the appropriate substrate, because of its relative ease of enzymatic hydrolysis to 5-N-acetyl-d-neuraminic acid and 5-bromoindoxyl. The latter is readily transformed to the insoluble, intensely coloured 5,5-dibromo-indigo. This precipitates and can be seen readily at the sites of enzymatic activity. The new substrate is of definitive advantage as it provides a simple and direct method for the demonstration of sialidases without the need of a coupling reaction.Abbreviations Neu5Ac 5-N-acetyl-d-neuraminic acid - BIN 5-bromo-indol-3-yl -ketoside ofN-acetyl-d-neuraminic acid     

Biochemical and immuno- and lectin-histochemical studies of solubility and retention of bone matrix proteins during EDTA demineralization

Summary The present study utilized biochemical and immuno-and lectin-histochemical methods to demonstrate solubility and retention of mineral-binding non-collagenous proteins in rat midshaft subperiosteal bone during EDTA demineralization. A monoclonal antibody (9-A-2) specific for chondroitin 4-sulphate and dermatan sulphate and wheat germ agglutinin (WGA) specific forN-acetyl-d-glucosamine,N-acetylneuraminic acid, andN-acetyl-d-galactosamine were used. Bone proteins were extracted from fresh unfixed or aldehyde-fixed specimens with a three step extraction procedure, 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl, followed by GdnCl. For comparison with the second extraction step, ethanolic trimethylammonium EDTA (ethanolic EDTA) was substituted for aqueous EDTA. Based on protein staining and Western blot analysis of SDS-polyacrylamide gel electrophoresis of each extract using 9-A-2 and WGA, retention of mineral-binding proteins extractable from fresh specimens with aqueous EDTA was greatly increased in tissue when ethanolic EDTA was used. Their retention was even greater with prior aldehyde fixation. Maximum retention with no detectable solubility of 9-A-2 and WGA reactive proteins was obtained after ethanolic EDTA extraction of aldehyde-fixed specimens, which concomitantly provided the strongest immuno- and lectin staining. These results indicate that this combined method dramatically improves retention of PGs and glycoproteins during demineralization of bone tissues and provides the best method for localizing these glycoconjugates.     

Enzymatic degradation and quantitative lectin labeling for characterizing glycoconjugates which act as lectin acceptors in cat submandibular gland

Summary Sites of binding of eight different lectins (LTA, UEA I, WGA, SBA, DBA, CON A, PNA, RCA I) to cat submandibular gland were studied after exposure of tissue sections to sialidase, -fucosidase, -galactosidase, -mannosidase, -N-acetylglucosaminidase. All lectins were affected by enzymatic predigestion and the labeling of individual lectins was highly dependent upon the glycosidase used to pretreat the sections. Glycoconjugates of demilunar, acinar and ductal cells exhibited a different composition of terminal sequences. For example, fucose proved to form the disaccharide fucose-galactose in demilunar and acinar cells, whereas it was present with the sequence fucose-N-acetyl-d-glucosamine in striated duct cells. Sialic acid participated both to the terminal sequence sialic acid-galactose and sialic acid-N-acetyl-d-galactosamine either in demilunar or in ductal cells. Lectin labeling combined with glycosidase digestion was also helpful in verifying the influence of neighbouring oligosaccharides on the affinity of lectins for the respective sugars.     

An efficient method for N-acetyl-d-neuraminic acid production using coupled bacterial cells with a safe temperature-induced system

N-Acetyl-d-neuraminic acid (Neu5Ac) is a precursor for producing many pharmaceutical drugs such as zanamivir which have been used in clinical trials to treat and prevent the infection with influenza virus, such as the avian influenza virus H5N1 and the current 2009 H1N1. Two recombinant Escherichia coli strains capable of expressing N-acetyl-d-glucosamine 2-epimerase and N-acetyl-d-neuraminic acid aldolase were constructed based on a highly efficient temperature-responsive expression system which is safe compared to chemical-induced systems and coupled in Neu5Ac production. Carbon sources were optimized for Neu5Ac production, and the concentration effects of carbon sources on the production were investigated. With 2,200 mM pyruvate as carbon source and substrate, 61.9 mM (19.1 g l⁻¹) Neu5Ac was produced from 200 mM N-acetyl-d-glucosamine (GlcNAc) in 36 h by the coupled cells. Our Neu5Ac biosynthetic process is favorable compared with natural product extraction, chemical synthesis, or even many other biocatalysis processes.     

Histochemical detection of sugar residues in the chick embryo mesonephros with lectin-horseradish peroxidase conjugates

Summary Fragments of mesonephros were taken from chick embryos and studied from the 4th to the 21st day of incubation. A battery of seven different horseradish peroxidase-labelled lectins was used to study the distribution of carbohydrate residues in glycoconjugates along the mesonephric nephron during the period of excretory activity and the period of involution. ConA and WGA reacted at every site of the nephron thus showing the ubiquitous presence of -D-mannose andN-acetyl-d-glucosamine. SBA was a good marker of the proximal tubule. Other lectins, such as PNA and LTA, reacted only for a short time at some sites during the considered period of incubation. The presence of sialic acid was detected in the podocytes, capillary wall and mesangial cells. From the 10th-11th day of incubation changes were noted in the proximal tubule as shown by PNA reactivity. This may be significant as regards the exact stage of incubation during which the involution of mesonephros begins.     

Characterization of raffinose synthase from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L. var. Nipponbare)

The putative raffinose synthase gene from rice was cloned and expressed in Escherichia coli. The enzyme displayed an optimum activity at 45°C and pH 7.0, and a sulfhydryl group was required for its activity. The enzyme was specific for galactinol and p-nitrophenyl-α-d-galactoside as galactosyl donors, and sucrose, lactose, 4−β-galactobiose, N-acetyl-d-lactosamine, trehalose and lacto-N-biose were recognized as galactosyl acceptors.