Microheterogeneity of the inner core region of yeast manno-protein

The inner core linkage region fragment from Saccharomyces cerevisiae mannan has been fractionated into 6 components and their structures have been analyzed. They form a family of homologous oligosaccharides (Man₁₂GNAc to Man₁₇GNAc) with 6 or 7 mannose units in α1→6 linkage attached to N-acetylglucosamine by a β1→4 linkage, and with different amounts of side chain mannose units attached by α1→2 and α1→3 linkage.     

Purification and characterization of β-xylosidase that is active for plant complex type N-glycans from tomato (Solanum lycopersicum): removal of core α1-3 mannosyl residue is prerequisite for hydrolysis of β1-2 xylosyl residue

In this study, we purified and characterized the β-xylosidase involved in the turnover of plant complex type N-glycans to homogeneity from mature red tomatoes. Purified β-xylosidase (β-Xyl’ase Le-1) gave a single band with molecular masses of 67 kDa on SDS-PAGE under a reducing condition and 60 kDa on gelfiltration, indicating that β-Xyl’ase Le-1 has a monomeric structure in plant cells. The N-terminal amino acid could not be identified owing to a chemical modification. When pyridylaminated (PA-) N-glycans were used as substrates, β-Xyl’ase Le-1 showed optimum activity at about pH 5 at 40 °C, suggesting that the enzyme functions in a rather acidic circumstance such as in the vacuole or cell wall. β-Xyl’ase Le-1 hydrolyzed the β1-2 xylosyl residue from Man₁Xyl₁GlcNAc₂-PA, Man₁Xyl₁Fuc₁GlcNAc₂-PA, and Man₂Xyl₁Fuc₁GlcNAc₂-PA, but not that from Man₃Xyl₁GlcNAc₂-PA or Man₃Xyl₁Fuc₁GlcNAc₂-PA, indicating that the α1-3 arm mannosyl residue exerts significant steric hindrance for the access of β-Xyl’ase Le-1 to the xylosyl residue, whereas the α1-3 fucosyl residue exerts little effect. These results suggest that the release of the β1-2 xylosyl residue by β-Xyl’ase Le-1 occurs at least after the removal the α-1,3-mannosyl residue in the core trimannosyl unit.     

Subcomponents C1q of the first component of guinea pig and mouse complement: Comparative study of their asparagine-linked sugar chains

Guinea pig and mouse C1q, subcomponents of the first component of complement, contained six asparagine-linked sugar chains on the C-terminal non-collagenous globular regions of each molecule. After N-acetylation and successive NaB³H₄-reduction of asparagine-linked sugar chains liberated by hydrazinolysis, their structure was analysed by sequential exoglycosidase digestion in combination with sugar composition analyses. The sugar chains of C1q molecules of both animals were very similar and composed of the biantennary complex type sugar chains with the following outer chains in various combinations: (± NeuNAcα → )Galß1 → GlcNAcß1 → and Galß1 → Galß1 → GlcNAcß1 →. These chain moieties were found to be linked to a common core structure of Manα1 → (Manα1 → )Manß1 → GlcNAcß1 → (Fucα1 → )GlcNAc.     

The molecular characterization of a novel GH38 α-mannosidase from the crenarchaeon Sulfolobus solfataricus revealed its ability in de-mannosylating glycoproteins

α-Mannosidases, important enzymes in the N-glycan processing and degradation in Eukaryotes, are frequently found in the genome of Bacteria and Archaea in which their function is still largely unknown. The α-mannosidase from the hyperthermophilic Crenarchaeon Sulfolobus solfataricus has been identified and purified from cellular extracts and its gene has been cloned and expressed in Escherichia coli. The gene, belonging to retaining GH38 mannosidases of the carbohydrate active enzyme classification, is abundantly expressed in this Archaeon. The purified α-mannosidase activity depends on a single Zn²⁺ ion per subunit is inhibited by swainsonine with an IC₅₀ of 0.2 mM. The molecular characterization of the native and recombinant enzyme, named Ssα-man, showed that it is highly specific for α-mannosides and α(1,2), α(1,3), and α(1,6)-d-mannobioses. In addition, the enzyme is able to demannosylate Man₃GlcNAc₂ and Man₇GlcNAc₂ oligosaccharides commonly found in N-glycosylated proteins. More interestingly, Ssα-man removes mannose residues from the glycosidic moiety of the bovine pancreatic ribonuclease B, suggesting that it could process mannosylated proteins also in vivo. This is the first evidence that archaeal glycosidases are involved in the direct modification of glycoproteins.     

Degradation of Misfolded Endoplasmic Reticulum Glycoproteins in Saccharomyces cerevisiae Is Determined by a Specific Oligosaccharide Structure

In Saccharomyces cerevisiae, transfer of N-linked oligosaccharides is immediately followed by trimming of ER-localized glycosidases. We analyzed the influence of specific oligosaccharide structures for degradation of misfolded carboxypeptidase Y (CPY). By studying the trimming reactions in vivo, we found that removal of the terminal α1,2 glucose and the first α1,3 glucose by glucosidase I and glucosidase II respectively, occurred rapidly, whereas mannose cleavage by mannosidase I was slow. Transport and maturation of correctly folded CPY was not dependent on oligosaccharide structure. However, degradation of misfolded CPY was dependent on specific trimming steps. Degradation of misfolded CPY with N-linked oligosaccharides containing glucose residues was less efficient compared with misfolded CPY bearing the correctly trimmed Man₈GlcNAc₂ oligosaccharide. Reduced rate of degradation was mainly observed for mis- folded CPY bearing Man₆GlcNAc₂, Man₇GlcNAc₂ and Man₉GlcNAc₂ oligosaccharides, whereas Man₈GlcNAc₂ and, to a lesser extent, Man₅GlcNAc₂ oligosaccharides supported degradation. These results suggest a role for the Man₈GlcNAc₂ oligosaccharide in the degradation process. They may indicate the presence of a Man₈GlcNAc₂-binding lectin involved in targeting of misfolded glycoproteins to degradation in S. cerevisiae.     

Substrate recognition of the catalytic α-subunit of glucosidase II from Schizosaccharomyces pombe

The recombinant catalytic α-subunit of N-glycan processing glucosidase II from Schizosaccharomyces pombe (SpGIIα) was produced in Escherichia coli. The recombinant SpGIIα exhibited quite low stability, with a reduction in activity to <40% after 2-days preservation at 4 °C, but the presence of 10% (v/v) glycerol prevented this loss of activity. SpGIIα, a member of the glycoside hydrolase family 31 (GH31), displayed the typical substrate specificity of GH31 α-glucosidases. The enzyme hydrolyzed not only α-(1→3)- but also α-(1→2)-, α-(1→4)-, and α-(1→6)-glucosidic linkages, and p-nitrophenyl α-glucoside. SpGIIα displayed most catalytic properties of glucosidase II. Hydrolytic activity of the terminal α-glucosidic residue of Glc₂Man₃-Dansyl was faster than that of Glc₁Man₃-Dansyl. This catalytic α-subunit also removed terminal glucose residues from native N-glycans (Glc₂Man₉GlcNAc₂ and Glc₁Man₉GlcNAc₂) although the activity was low.     

Specificity in the enzymic transfer of sialic acid to the oligosaccharide branches of BI- and triantennary glycopeptides of α1-acid glycoprotein

Partial $\text{in}\text{vitro}$ sialylation of biantennary and triantennary glycopeptides of α₁-acid glycoprotein using colostrum β-galactosideα(2→6) sialyltransferase followed by high resolution ¹H-NMR spectroscopic analysis of the isolated products enabled the assignment of the $\text{Galβ(1→4)GlcNAcβ(1→2)Man}\text{α(1→3)}\text{Man}$ branch as the most preferred substrate site for sialic acid attachment. The $\text{Galβ(1→4)GlcNAcβ(1→2)Man}\text{α(1→6)}\text{Man}$ branch appeared to be much less preferred and the Galβ(1→4)GlcNAcβ(1→4)Manα(1→3)Man sequence of triantennary structures was of intermediate preference for the sialyltransferase. The specificity of the β-galactoside α(2→6) sialyltransferase is thus shown to extend to structural features beyond the terminal $\text{N}$-acetyllactosamine units on the oligosaccharide chains of serum glycoproteins.     

The carbohydrate units of ovalbumin: Complete structures of three glycopeptides

Three glycopeptides, obtained in quantity from ovalbumin by exhaustive digestion with Pronase and purified by ion-exchange chromatography and gel filtration, had mannose-2-acetamido-2-deoxyglucose-aspartic acid ratios of 5:4:1, 6:2:1, and 5:2:1. The structures of the glycopeptides have been investigated by sequential digestion with purified exo-glycosidases, Smith degradation, and selective acetolysis, and by methylation analysis of the glycopeptides and their degradation products. The resulting data indicated the structures to be α-d-Manp-(1→6)-[α-d- Manp-(1→3)]-α-d-Manp-(1→6)-[β-d-GlcNAcp-(1→4)]-[β-d-GlcNAcp-(1→2)-α-d- Manp-(1→3)]-β-d-Manp-(1→4)-β-d-GlcNAcp-(1→4)-β-d-GlcNAcp→Asn, α-d- Manp-(1→6)-[α-d-Manp-(1→3)]-α-d-Manp-(1→6)-[α-d-Manp-(1→2)-α-d-Manp- (1→3)]-β-d-Manp-(1→4)-β-d-GlcNAcp-(1→4)-β-d-GlcNAcp→Asn, and α-d-Manp- (1→6)-[α-d-Manp-(1→3)]-α-d-Manp-(1→6)-[α-d-Manp-(1→3)]-β-d-Manp-(1→4)- β-d-GlcNAcp-(1→4)-β-d-GlcNAcp→Asn. The glycopeptides had a common-core structure consisting of five mannose and two hexosamine residues, but the two larger glycopeptides were not homologous.     

The nature of mannose-containing material which accumulates in cultured fibroblasts from patients with mannosidosis

Fibroblasts from a patient with mannosidosis were grown in a medium containing a radioactive monosaccharide (D[U-¹⁴C]mannose or $\text{N-}\text{acetyl}\text{-}\text{D}\text{-[1-14}\text{C}\text{]-}\text{glucosamine}$). An accumulation of radioactive material was observed. It was possible to prevent the accumulation to a certain degree by the addition of human liver α-D-mannosidase to the fibroblast medium. After six days of fibroblast culture the majority of the accumulated material had a molecular weight in the oligosaccharide range and was stationary during high-voltage electropresis. Paper chromatography of the stationary material separated three radioactive compounds with the same chromatographic mobilities as the oligosaccharides $\text{α-}\text{D}\text{-}\text{Man}\text{-(1 → 3)-β-}\text{D}\text{-}\text{Man}\text{-(1 → 4)-}\text{D}\text{-}\text{GlcNAc}\text{(I), α-}\text{D}\text{-}\text{Man}\text{-(1 → 2)- α-}\text{D}\text{-}\text{Man}\text{-(1 → 3)-β-}\text{D}\text{-}\text{Man}\text{-(1 → 4)-}\text{GlcNAc}$ (II), and $\text{α-}\text{D}\text{-}\text{Man}\text{-(1 → 2)-α-}\text{D}\text{-}\text{Man}\text{- (1 → 2)-α-}\text{D}\text{-}\text{Man}\text{-(1 → 3)-β-}\text{D}\text{-}\text{Man}\text{-(1 → 4)-}\text{GlcNAc}$ (III) previously isolated from the urine of patients with mannosidosis. Degradation of the three radioactive compounds with jack bean α-mannosidase gave D-mannose and a disaccharide (containing D-mannose and $\text{N-}\text{acetyl}\text{-}\text{D}\text{-}\text{glucosamine}$). Thus the three main compounds observed in the fibroblast from patients with mannosidosis are most probably identical to the oligosaccharides I–III.     

Conversion of α1,2-mannosidase E-I from Candida albicans to α1,2-mannosidase E-II by limited proteolysis

Previous studies demonstrated the presence in Candida albicans ATCC 26555 of two soluble α1,2-mannosidases: E-I and E-II. In contrast, in the C. albicans CAI-4 mutant only E-I was detected and it could be processed by a membrane-bound proteolytic activity from the ATCC 26555 strain, generating an active 43 kDa polypeptide. Here, α1,2-mannosidase E-I from strain ATCC 26555 was purified by conventional methods of protein isolation and affinity chromatography in Concanavalin A-Sepharose 4B. Analytical electrophoresis of the purified enzyme revealed two polypeptides of 52 and 23 kDa, the former being responsible for enzyme activity as revealed by zymogram analysis. Time course proteolysis with an aspartyl protease from Aspergillus saitoi, converted α1,2-mannosidase E-I into an active polypeptide of 43 kDa which trimmed Man₉GlcNAc₂, generating Man₈GlcNAc₂ isomer B and mannose. Trimming was inhibited preferentially by 1-deoxymannojirimycin. Both, the molecular mass and the enzyme properties of the proteolytic product were identical to those described for α1,2-mannosidase E-II therefore supporting the notion that E-I is the precursor of E-II.     

Biosynthesis of mannose-containing lipid-linked oligosaccharides by solubilized enzyme preparation from cultured soybean cells

Glycosyl transferases that participate in the assembly of the lipid-linked oligosaccharide intermediates were solubilized from cultured soybean cells using 0.3% Nonidet P-40 (NP-40) in the presence of 10% glycerol. The solubilized enzyme preparation was reasonably stable and 50% of the activity still remained after storage at −10°C for 1 month. The solubilized enzyme synthesized [¹⁴C]Man₃GlcNAc₂-pyrophosphoryl-polyprenol and [¹⁴C]Man₅GlcNAc₂-pyrophosphoryl-polyprenol when incubated with GDP-[¹⁴C]mannose plus a partially purified acceptor lipid isolated from calf liver. The formation of these lipid-linked oligosaccharides did not require the addition of dolichyl-phosphate or metal ions. In fact, the addition of 5 to 10 millimolar ethylenediaminetetraacetate stimulated the incorporation of mannose into lipid-linked oligosaccharides 2- to 3-fold. Since little or no dolichyl-phosphoryl-mannose is formed in the presence of ethylenediaminetetraacetate, the results suggest that the mannosyl residues added to form Man₃GlcNAc₂-lipid and Man₅GlcNAc₂-lipid come directly from GDP-mannose without the participation of dolichyl-phosphoryl-mannose. On the other hand, the formation of significant amounts of Man₆GlcNAc₂-lipid, Man₇GlcNAc₂-lipid, and Man₈GlcNAc₂-lipid occurred when the above incubations were supplemented with dolichyl-phosphate and metal ions. Based on various time course studies and supplementation studies with various additions, it appears likely that the first five mannose residues to form Man₅GlcNAc₂-lipid come directly from GDP-mannose, whereas other mannose units to form larger oligosaccharide-lipids come from dolichyl-phosphoryl-mannose.     

The amino acid sequence of toxin V from Anemonia sulcata

Preparations of the β-galactoside-binding lectin of bovine heart have been shown to stimulate $\text{in vitro}$ the sialylation of the oligosaccharide Ga1β1→4G1cNAc and asialo-α₁-acid glycoprotein by bovine colostrum β-D-galactoside α2→6 sialyltransferase. Kinetic data revealed that in the presence of lectin the K_m values for Ga1β1→4G1cNAc and CMP-NeuAc were reduced from 25.0 to 11.6 mM and from 0.42 to 0.19 mM respectively, but the K_m for asialo-α₁-acid glycoprotein and the V_max values for all three substrates were little affected. Stimulation by the lectin was partially inhibited by Fucα1→2Ga1β1→4G1cNAc. This, together with the effects of certain plant lectins, suggests that the stimulation of sialytransferase may be mediated through the carbohydrate-binding properties of the lectin.     

Lack of the evidence for the enzymatic catabolism of Man1GlcNAc2 in Saccharomyces cerevisiae

In the cytosol of Saccharomyces cerevisiae, most of the free N-glycans (FNGs) are generated from misfolded glycoproteins by the action of the cytoplasmic peptide: N-glycanase (Png1). A cytosol/vacuole α-mannosidase, Ams1, then trims the FNGs to eventually form a trisaccharide composed of Manβ1,4GlcNAc β1,4GlcNAc (Man₁GlcNAc₂). Whether or not the resulting Man₁GlcNAc₂ is enzymatically degraded further, however, is currently unknown. The objective of this study was to unveil the fate of Man₁GlcNAc₂ in S. cerevisiae. Quantitative analyses of the FNGs revealed a steady increase in the amount of Man₁GlcNAc₂ produced in the post-diauxic and stationary phases, suggesting that this trisaccharide is not catabolized during this period. Inoculation of the stationary phase cells into fresh medium resulted in a reduction in the levels of Man₁GlcNAc₂. However, this reduction was caused by its dilution due to cell division in the fresh medium. Our results thus indicate that Man₁GlcNAc₂ is not enzymatically catabolized in S. cerevisiae.     

Protease-Deficient Saccharomyces cerevisiae Strains for the Synthesis of Human-Compatible Glycoproteins

Saccharomyces cerevisiae strains engineered previously to produce proteins with mammalian high mannose structures showed severe growth defects and decreased protein productivity. In strain YAB101, derived from one of these strains by a mutagenesis technique based on the disparity theory of evolution, these undesirable phenotypes were alleviated. Here we describe further engineering of YAB101 with the aim of synthesizing heterologous glycoproteins with Man₅GlcNAc₂, an intermediate for the mammalian hybrid and complex type oligosaccharides. About 60% conversion of Man₈GlcNAc₂ to Man₅GlcNAc₂ was observed after integration of Aspergillus saitoi α-1,2-mannosidase fused to the transmembrane domain of S. cerevisiae Och1. To obtain a higher yield of the target protein, a protease-deficient version of this strain was generated by disruption of PEP4 and PRB1, resulting in YAB101-4. Inactivation of these vacuolar proteases enhanced the secretion of human interferon-β by approximately 10-fold.     

NovelN-linked oligo-mannose type oligosaccharides containing an α-d-galactofuranosyl linkage found in α-d-galactosidase fromAspergillus niger

Structures of oligosaccharides fromAspergillus niger -d-galactosidase [EC 3.2.1.22] were studied. Purified -d-galactosidase was treated withN-glycosidase F, and six kinds of oligosaccharides were isolated by gel chromatography and anion-exchange chromatography. The structures of the oligosaccharides were determined by¹H-NMR and compositional analysis to be Man₅GlcNAc₂, Man₆GlcNAc₂, Man₉GlcNAc₂, GlcMan₉GlcNAc₂, GalMan₄GlcNAc₂ and GalMan₅GlcNAc₂. From mild acid hydrolysis, methylation analysis and ROESY spectral analysis, it was ascertained that the galactosyl residue in two oligosaccharides was in the furanose form and was bound to mannose at the nonreducing end with an 1–2 linkage (GalfMan₄GlcNAc₂ and GalfMan₅GlcNAc₂).     

A glucose/mannose binding lectin from litchi (Litchi chinensis) seeds: Biochemical and biophysical characterizations

BackgroundLectins are highly important biomolecules to study several biological processes. A novel α-D-glucose/mannose specific lectin was isolated from the seeds of litchi fruits (Litchi chinensis) and its various biophysical and biochemical properties were studied.MethodsPurification was done by successive Sephadex G 100 and Con A-Sepharose 4B affinity chromatography. SDS-PAGE, Surface Plasmon Resonance (SPR), steady state absorbance, fluorescence, time-correlated single-photon counting, circular dichroism and antibiofilm activity by measuring total protein estimation and azocasein degradation assay have been performed.ResultsThe purified lectin is a homodimer of molecular mass ~ 54 kDa. The amount of lectin required for hemagglutination of normal human O erythrocytes was 6.72 µg/ml. Among the saccharides tested, Man-α-(1,6)-Man was found to be the most potent inhibitor (0.01 mM) determined by hemagglutination inhibition assay. Steady state and time resolved fluorescence measurements revealed that litchi lectin formed ground state complex with maltose (K_a=4.9 (±0.2)×10⁴ M^?1), which indicated static quenching (Stern-Volmer (SV) constant K_sv=4.6 (±0.2)×10⁴ M^?1). CD measurements demonstrated that litchi lectin showed no overall conformational change during the binding process with maltose. The lectin showed antibiofilm activity against Pseudomonus aeruginosa.ConclusionsA novel homodimeric lectin has been purified from the seeds of litchi fruits (Litchi chinensis) having specificity for α-d-glucose/mannose. The thermodynamics and conformational aspects of its interaction with maltose have been studied in detail. The antibiofilm activity of this lectin towards Pseudomonus aeruginosa has been explored.General significanceThe newly identified litchi lectin is highly specific for α-d-glucose/mannose with an important antibiofilm activity towards Pseudomonus aeruginosa.     

Cation-independent Mannose 6-Phosphate Receptor: A COMPOSITE OF DISTINCT PHOSPHOMANNOSYL BINDING SITES*

The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR), which contains multiple mannose 6-phosphate (Man-6-P) binding sites that map to domains 3, 5, and 9 within its 15-domain extracytoplasmic region, functions as an efficient carrier of Man-6-P-containing lysosomal enzymes. To determine the types of phosphorylated N-glycans recognized by each of the three carbohydrate binding sites of the CI-MPR, a phosphorylated glycan microarray was probed with truncated forms of the CI-MPR. Surface plasmon resonance analyses using lysosomal enzymes with defined N-glycans were performed to evaluate whether multiple domains are needed to form a stable, high affinity carbohydrate binding pocket. Like domain 3, adjacent domains increase the affinity of domain 5 for phosphomannosyl residues, with domain 5 exhibiting ∼60-fold higher affinity for lysosomal enzymes containing the phosphodiester Man-P-GlcNAc when in the context of a construct encoding domains 5–9. In contrast, domain 9 does not require additional domains for high affinity binding. The three sites differ in their glycan specificity, with only domain 5 being capable of recognizing Man-P-GlcNAc. In addition, domain 9, unlike domains 1–3, interacts with Man₈GlcNAc₂ and Man₉GlcNAc₂ oligosaccharides containing a single phosphomonoester. Together, these data indicate that the assembly of three unique carbohydrate binding sites allows the CI-MPR to interact with the structurally diverse phosphorylated N-glycans it encounters on newly synthesized lysosomal enzymes.     

Identification of high-mannose and multiantennary complex-type <Emphasis Type="Italic">N</Emphasis>-linked glycans containing α-galactose epitopes from Nurse shark IgM heavy chain

MALDI-TOF mass spectrometry, negative ion nano-electrospray MS/MS and exoglycosidase digestion were used to identify 36 N-linked glycans from 19S IgM heavy chain derived from the nurse shark (Ginglymostoma cirratum). The major glycan was the high-mannose compound, Man₆GlcNAc₂ accompanied by small amounts of Man₅GlcNAc₂, Man₇GlcNAc₂ and Man₈GlcNAc₂. Bi- and tri-antennary (isomer with a branched 3-antenna) complex-type glycans were also abundant, most contained a bisecting GlcNAc residue (β1→4-linked to the central mannose) and with varying numbers of α-galactose residues capping the antennae. Small amounts of monosialylated glycans were also found. This appears to be the first comprehensive study of glycosylation in this species of animal. The glycosylation pattern has implications for the mechanism of activation of the complement system by nurse shark IgM.     

Immunochemical studies on the combining site of the d-galactose/N-acetyl-d-galactosamine specific lectin from Erythrina cristagalli seeds

The combining site of the Erythrina cristagalli lectin was studied by quantitative precipitin and precipitin inhibition assays. The lectin precipitated best with two fractions of a precursor human ovarian cyst blood group substance with I and i activities. A₁, A₂, B, H, Le^a, and Le^b blood group substances precipitated poorly to moderately and substances of the same blood group activity precipitated to varying extents. These differences are attributable to heterogeneity resulting from incomplete biosynthesis of carbohydrate chains. Specific precipitates with the poorly reactive blood group substances were found to be more soluble than those reacting strongly. Precipitation was minimally affected by EDTA or divalent cations. Among the monosaccharides and glycosides tested for inhibition of precipitation, p-nitrophenyl βdGal was most active and was 10 times more active than methyl βdGal, indicating involvement of hydrophobic contacts in site specificity. Methyl αdGalNAc, p-nitrophenyl αdGalNAc, methyl αdGal, N-acetyl-d-galactosamine, p-nitrophenyl αdGal, methyl βdGal, and p-nitrophenyl βdGalNAc were progressively less active than p-nitrophenyl βdGal. The best disaccharide inhibitor dGalβ1 → 4dGlcNAc was 7.5 times more potent than p-nitrophenyl βdGal. A tetraantennary and triantennary oligosaccharide containing four and three dGalβ1 → 4dGlcNAcβ1 → branches, respectively, were, because of cooperative binding effects, 1.6 and 2.5 times more active than the bi- and monoantennary oligosaccharides, respectively. dGalβ1 → 4dGlcNAcβ1 → 6dGal and dGalβ1 → 4dGlcNAcβ1 → 2dMan had the same activity, being 1.5 times more active than dGalβ1 → 4dGlcNAc, which was 2.6 and 8.5 times more active than dGalβ1 → 3dGlcNAc and dGalβ1 → 3dGlc, respectively. Substitutions by N-acetyl-d-galactos-amine or l-fucose on the d-galactose of inhibitory compounds blocked activity. These results suggest that a hydrophobic interaction with the subterminal sugar is important in the binding and that the specificity of the lectin combining site involves a terminal dGalβ1 → 4dGlcNAc and the β linkage of a third sugar.