Α-linked Galactose Research Articles

The structure of a family 110 glycoside hydrolase provides insight into the hydrolysis of α-1,3-galactosidic linkages in λ-carrageenan and blood group antigens

α-Linked galactose is a common carbohydrate motif in nature that is processed by a variety of glycoside hydrolases from different families. Terminal Galα1–3Gal motifs are found as a defining feature of different blood group and tissue antigens, as well as the building block of the marine algal galactan λ-carrageenan. The blood group B antigen and linear α-Gal epitope can be processed by glycoside hydrolases in family GH110, whereas the presence of genes encoding GH110 enzymes in polysaccharide utilization loci from marine bacteria suggests a role in processing λ-carrageenan. However, the structure–function relationships underpinning the α-1,3-galactosidase activity within family GH110 remain unknown. Here we focus on a GH110 enzyme (PdGH110B) from the carrageenolytic marine bacterium Pseudoalteromonas distincta U2A. We showed that the enzyme was active on Galα1–3Gal but not the blood group B antigen. X-ray crystal structures in complex with galactose and unhydrolyzed Galα1–3Gal revealed the parallel β-helix fold of the enzyme and the structural basis of its inverting catalytic mechanism. Moreover, an examination of the active site reveals likely adaptations that allow accommodation of fucose in blood group B active GH110 enzymes or, in the case of PdGH110, accommodation of the sulfate groups found on λ-carrageenan. Overall, this work provides insight into the first member of a predominantly marine clade of GH110 enzymes while also illuminating the structural basis of α-1,3-galactoside processing by the family as a whole.

Nicotiana benthamiana α-galactosidase A1.1 can functionally complement human α-galactosidase A deficiency associated with Fabry disease

α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall-associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activity-based probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-d-galactopyranoside substrate (Km = 0.17 mm) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management.

Synthetic avenues towards a tetrasaccharide related to Streptococcus pneumonia of serotype 6A.

Streptococcus pneumonia (SPn) is a Gram-positive bacterium which causes life threatening diseases. The bacteria protect themselves against non-specific host defence by an external polysaccharide (PS) capsule which bears a repeating unit, α-D-Galp(1->3)-α-D-Glcp(1->3)-α-L-Rhap(1->3)-D-Rib (SPn 6A). A closer look at the structure reveals the presence of α-linked galactose and glucose residues. The synthesis of these 1,2-cis glycosidic linkages are considered challenging particularly in the context of a one-pot oligosaccharide synthesis. We have synthesized the aforesaid tetrasaccharide (SPn 6A) based on both stepwise and sequential one-pot glycosylation reactions using easily accessible common building blocks; eventually similar overall yields were obtained in both cases.

Carrageenans and carrageenases: versatile polysaccharides and promising marine enzymes

Carrageenans are sulfated polysaccharides isolated from marine red algae that share a common backbone of D-galactose alternately linked by α(1,3) and β(1,4) glycosidic linkages. They are classified based on the number and the position of the sulfate ester groups and the occurrence of a 3,6 anhydro-ring in the α-linked galactose. Accordingly the three most commercially exploited carrageenans are κ-, ι-, and λ-carrageenans. Because of their biocompatibility, exceptional physicochemical features and emulsifying, thickening, gelling and stabilizing abilities, they have found several industrial application, especially in food, pharmaceutical and cosmetic industries. Moreover, carrageenans can be degraded into lower molecular weight oligosaccharides, which have been reported to have promising pharmacological properties and potential therapeutic applications. Enzymes which degrade carrageenans are called carrageenases and are produced only by marine bacterial species. These enzymes all are endohydrolases that hydrolyze the internal β 1,4 linkages in carrageenans and produce a series of homologous even-numbered oligosaccharides with various biological and physiological activities including anti-tumor, anti-inflammation, anti-viral, anti-coagulation, etc. Carrageenase enzymes have also other applications related to the biomedical field, bioethanol production, prevention of red algal bloom, obtaining algal protoplasts, etc. In the first part of this review, general information regarding structure, physicochemical properties, biological activities and potential applications of carrageenans is summarized. The second part deals with research and development works on some aspects of carrageenase enzymes like the source, characterization, the kinetics and biochemical properties and their applications in various industries.

Core Richness of N-Glycans of Caenorhabditis elegans: A Case Study on Chemical and Enzymatic Release.

Despite years of research, the glycome of the model nematode Caenorhabditis elegans is still not fully understood. Certainly, data over the years have indicated that this organism synthesizes unusual N-glycans with a range of galactose and fucose modifications on the Man2–3GlcNAc2 core region. Previously, up to four fucose residues were detected on its N-glycans, despite these lacking the fucosylated antennae typical of many other eukaryotes; some of these fucose residues are capped with hexose residues as shown by the studies of us and others. There have, though, been contrasting reports regarding the maximal number of fucose substitutions in C. elegans, which in part may be due to different methodological approaches, including use of either peptide:N-glycosidases F and A (PNGase F and A) or anhydrous hydrazine to cleave the N-glycans from glycopeptides. Here we compare the use of hydrazine with that of a new enzyme (rice PNGase Ar) and show that both enable release of glycans with more sugar residues on the proximal GlcNAc than previously resolved. By use of exoglycosidase sequencing, in conjunction with high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS), we now reveal that actually up to five fucose residues modify the core region of C. elegans N-glycans and that the α1,3-fucose on the reducing terminus can be substituted by an α-linked galactose. Thus, traditional PNGase F and A release may be insufficient for release of the more highly core-modified N-glycans, especially those occurring in C. elegans, but novel enzymes can compete against chemical methods in terms of safety, ease of cleanup, and quality of resulting glycomic data.

Identification and Characterization of Two Novel Alpha-D-Galactosidases from Pedobacter heparinus.

Two putative α-D-galactosidases (α-GALs) belonging to glycosyl hydrolase family 27, and originating from the rather unexplored bacterial strain Pedobacter heparinus, were cloned and biochemically characterized. The recombinant enzymes designated as PhAGal729 and PhAGal2920 showed comparable biochemical properties: the optimum pH values were determined to be pH 5.0 and 5.5, and temperature optima lay between 30°C and 37°C, respectively. Both α-GALs were not dependent on the presence of divalent metal ions, and the addition of EDTA had no influence on enzymatic activity. The activity of both enzymes substantially increased in the presence of Fe3(+) ions. Both enzymes were inhibited by sodium dodecyl sulfate (SDS) and urea. α-GALs from P. heparinus were highly specific in hydrolyzing glycosides with α-1,2/3/4 or α-1,6-linked galactose to other sugars, whereas other glycosides such as α-linked N-acetylgalactosamine, N-acetylglucosamine or glucose residues were not released. Nevertheless, neither PhAGal729 nor PhAGal2920 were able to remove α-linked galactose epitopes from native human erythrocytes. The facile expression and purification procedures in combination with wide substrate specificities make α-GALs from P. heparinus potential candidates for applications in analytical research, and food- and biotechnology.

Structural studies of the exopolysaccharide from Lactobacillus plantarum C88 using NMR spectroscopy and the program CASPER

Some lactic acid bacteria, such as those of the Lactobacillus genus, have the ability to produce exopolysaccharides (EPSs) that confer favorable physicochemical properties to food and/or beneficial physiological effects on human health. In particular, the EPS of Lactobacillus plantarum C88 has recently demonstrated in vitro antioxidant activity and, herein, its structure has been investigated using NMR spectroscopy and the computer program CASPER (Computer Assisted Spectrum Evaluation of Regular polysaccharides). The pentasaccharide repeating unit of the O-deacetylated EPS consists of a trisaccharide backbone, →4)-α-d-Galp-(1→2)-α-d-Glcp-(1→3)-β-d-Glcp-(1→, with terminal d-Glc and d-Gal residues (1.0 and 0.8equiv per repeating unit, respectively) extending from O3 and O6, respectively, of the →4)-α-d-Galp-(1→ residue. In the native EPS an O-acetyl group is present, 0.85equiv per repeating unit, at O2 of the α-linked galactose residue; thus the repeating unit of the EPS has the following structure: →4)[β-d-Glcp-(1→3)][β-d-Galp-(1→6)]α-d-Galp2Ac-(1→2)-α-d-Glcp-(1→3)-β-d-Glcp-(1→. These structural features, and the chain length (∼103 repeating units on average, determined in a previous study), are expected to play an important role in defining the physicochemical properties of the polymer.

Plasma anti-α-galactoside antibody binds to serine- and threonine-rich peptide sequence of apo(a) subunit in Lp(a).

Lipoprotein(a) immune complexes [Lp(a) IC] of varying particle density obtained by ultracentrifugation of plasma from normal healthy donors were markedly dominated by IgG. Lp(a) and immunoglobulins were liberated from plasma Lp(a) IC by treatment with melibiose, a sugar specific for circulating anti-α-galactoside antibody (anti-Gal). Upon incubation with plasma lipoprotein fraction anti-Gal but not the α-glucoside-specific antibody from human plasma formed de novo IC with Lp(a). Binding of Lp(a) sugar-reversibly enhanced the fluorescence of FITC-labeled anti-Gal as did binding of α-galactoside-containing glycoproteins. This effect apparently due to conformational shift in the Fc region of the antibody was also produced by apo(a) subunit separated from Lp(a) and de-O-glycosylated apo(a) but not by any other plasma lipoproteins or by Lp(a) pre-incubated with the O-glycan-specific lectin jacalin. O-Glycans and their terminal sialic acid moieties in apo(a) of circulating Lp(a)-anti-Gal IC, in contrast to those in pure Lp(a), were inaccessible to jacalin and anion exchange resin, respectively. Unlike other plasma lipoproteins, Lp(a) inhibited Griffonia simplicifolia isolectin B4 which also accommodates serine- and threonine-rich peptide sequence (STPS) as surrogate ligand to α-galactosides at its binding site. Results suggest that anti-Gal recognizes STPS in the O-glycan-rich regions of apo(a) subunit in Lp(a) which contains no α-linked galactose.

The Molecular Basis for Recognition of CD1d/α-Galactosylceramide by a Human Non-Vα24 T Cell Receptor

Author SummaryCertain lineages of T cells can recognize lipids as stimulatory antigens when presented in the context of CD1 molecules. We know how most Natural Killer T (NKT) cells react with this unusual ligand because they use a single invariant T cell receptor (TCR) alpha chain to do the job. NKT cells place particular emphasis on their CDR3α and CDR2β loops in recognition of antigen—these complementarity determining regions (CDRs) are the hypervariable parts of the TCR that “complement” an antigen's shape. How do these other T cells recognize closely related yet distinct lipid antigens? Here we show that human CD1d-restricted T cells, typically called Vα24− T cells due to their use of diverse Vα domains in their TCRs, use similar molecular strategies to respond to lipid antigens presented by CD1d. To this end we present a 2.5 Å complex structure of a Vα24− TCR complexed with CD1d presenting the protypical lipid, α-galactosylceramide (αGalCer). The TCR examined in this study notably shifts its binding slightly, placing more emphasis on the interaction with the CDR1α loop as revealed through alanine scanning mutagenesis. This shift explains the inability of these T cells to respond to lipids that vary at this site of contact (the 4'OH), like the related α-linked glucosylceramide. These results provide a molecular basis for the fine-specificity of different CD1d-restricted T cell lineages.

Functional Analysis of Family GH36 α-Galactosidases from Ruminococcus gnavus E1: Insights into the Metabolism of a Plant Oligosaccharide by a Human Gut Symbiont

Ruminococcus gnavus belongs to the 57 most common species present in 90% of individuals. Previously, we identified an α-galactosidase (Aga1) belonging to glycoside hydrolase (GH) family 36 from R. gnavus E1 (M. Aguilera, H. Rakotoarivonina, A. Brutus, T. Giardina, G. Simon, and M. Fons, Res. Microbiol. 163:14-21, 2012). Here, we identified a novel GH36-encoding gene from the same strain and termed it aga2. Although aga1 showed a very simple genetic organization, aga2 is part of an operon of unique structure, including genes putatively encoding a regulator, a GH13, two phosphotransferase system (PTS) sequences, and a GH32, probably involved in extracellular and intracellular sucrose assimilation. The 727-amino-acid (aa) deduced Aga2 protein shares approximately 45% identity with Aga1. Both Aga1 and Aga2 expressed in Escherichia coli showed strict specificity for α-linked galactose. Both enzymes were active on natural substrates such as melibiose, raffinose, and stachyose. Aga1 and Aga2 occurred as homotetramers in solution, as shown by analytical ultracentrifugation. Modeling of Aga1 and Aga2 identified key amino acids which may be involved in substrate specificity and stabilization of the α-linked galactoside substrates within the active site. Furthermore, Aga1 and Aga2 were both able to perform transglycosylation reactions with α-(1,6) regioselectivity, leading to the formation of product structures up to [Hex](12) and [Hex](8), respectively. We suggest that Aga1 and Aga2 play essential roles in the metabolism of dietary oligosaccharides and could be used for the design of galacto-oligosaccharide (GOS) prebiotics, known to selectively modulate the beneficial gut microbiota.

Cross‐reactive antigens for natural IgE antibodies: allergens with the potential to transform a dormant to an active allergic response?

Cross‐reactive antigens for natural IgE antibodies: allergens with the potential to transform a dormant to an active allergic response?

Balanced on One Foot

Cd1 proteins bind lipid antigens and present them to T cells to initiate immune responses. Cd1d presents glycolipids with specific α-linked sugars to the natural killer T cells (NKT) for recognition by their T cell receptor (TCR) complex. Borg et al . report the crystal structure of Cd1 bound to α-galactosylceramide (α-GalCer) with the NKT TCR. They find that the TCR interaction is unlike that of TCRs with the peptide-presenting major histocompatibility proteins (pMHCs). In the case of the pMHC-TCR interaction, the TCR α and β chains both contact the pMHC (see Moody), revealing two binding site "footprints" on the surface of the pMHC. The crystal structures reported by Borg et al . show that NKT TCR only binds to the Cd1d-α-GalCer by the α subunit, with the β subunit showing limited interaction and mostly hanging over the side of Cd1d. Moody describes the two structures as the TCR standing with both feet contacting pMHC, whereas NKT TCR appears as a pirouette balanced on one foot at the Cd1d surface. The structure also shows that the α-linked galactose lies flat, optimizing the interaction surface between Cd1d and the NKT TCR, which may provide a basis for the selective recognition of α-linked sugars. This study reinforces the notion that glycolipid-antigen recognition is achieved through molecular mechanisms different from those for peptide-antigen recognition and cautions against casting the Cd1 lipid-presenting proteins as MHC-like. N. A. Borg, K. S. Wun, L. Kjer-Nielsen, M. C. J. Wilce, D. G. Pellicci, R. Koh, G. S. Besra, M. Bharadwaj, D. I. Godfrey, J. McCluskey, J. Rossjohn, CD1d-lipid-antigen recognition by the semi-invariant NKT T-cell receptor. Nature 448 , 44-49 (2007). [PubMed] D. B. Moody, How a T cell sees sugar. Nature 448 , 36-37 (2007). [PubMed]

B to O erythrocyte conversion by the recombinant α-galactosidasc

Human group O red blood cells have great benefit in specialized transfusion areas such as armed conflict and natural calamity. The group B antigen differs structurally from group O antigen only by the addition of one terminal alpha-linked galactose residue. In this study we aimed to remove the terminal galactose from group B red blood cell to get group O red blood cell. alpha-galactosidase cDNA was cloned by RT-PCR from Catimor coffee beans grown on Hainan Island of China. The vector for alpha-galactosidase cDNA expression was constructed and transferred into Pichia pastoris cells by electroporation. The transgenic cells were cloned by fermentation and the recombinant alpha-galactosidase was purified by ion exchange chromatography. After studying the biochemical characters of alpha-galactosidase, we have used it in converting human erythrocytes from group B to group O. The purity of recombinant alpha-galactosidase was higher than 96%, which was thought to be suitable for the use of blood conversion. Enzymatically converted human group O red blood cells (ECHORBC) exhibited membrane integrity, metabolic integrity, normal cell deformation and morphology. There were no coagulation between ECHORBC and any group of human blood. The ECHORBC will keep normal structure and function for a period of 21 days at 4 degrees C in monoammoniumphosphate nutrient solution. Experiments with Rhesus monkeys and gibbons showed that transfusion of enzymatically converted erythrocytes was safe. ECHORBC can be easily obtained from group B red blood cell by alpha-galactosidase digestion. This study suggests that ECHORBC could be transfused to patients safely and efficiently.

Automated Synthesis of the Tumor-Associated Carbohydrate Antigens Gb-3 and Globo-H: Incorporation of α-Galactosidic Linkages

The Globo series of tumor-associated antigens are glycosphingolipids implicated in several cancer types. These oligosaccharides contain an α-linked galactose. Reported are methods to efficiently install cis-galactosidic linkages by automated solid-phase oligosaccharide synthesis. The complex tumor-associated oligosaccharides Globo-H and Gb-3 were assembled in about 1 day using the new approach that extends the scope of automated oligosaccharide synthesis to structurally more challenging carbohydrates.

Microanalysis of N-linked oligosaccharides in a glycoprotein by capillary liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry

We have studied rapid and simple sugar mapping using liquid chromatography/electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column. The oligosaccharide mixture was separated on the basis of the sequence, branching structure, and linkage, and each oligosaccharide was characterized based on its molecular mass. In this study we demonstrated the usefulness of capillary LC/MS (CapLC/MS) and capillary liquid chromatography/tandem mass spectrometry (CapLC/MS/MS) as sensitive means for accomplishing the structural analysis of oligosaccharides in a low-abundance glycoprotein. The carbohydrate heterogeneity and molecular mass information of each oligosaccharide can be readily obtained from CapLC/MS of a small amount of glycoprotein. CapLC/MS/MS provided b-ion series, which is informative with regard to monosaccharide sequence. Exoglycosidase digestion followed by CapLC/MS elucidated a carbohydrate residue linkage. Using this method, we characterized N-linked oligosaccharides in hepatocyte growth factor produced in mouse myeloma NS0 cells as the complex-type bi-, tri-, and tetraantennary terminated with N-glycolylneuraminic acids and α-linked galactose residues. Sugar mapping with CapLC/MS and CapLC/MS/MS is useful for monitoring glycosylation patterns and for structural analysis of carbohydrates in a low-abundance glycoprotein and thus will become a powerful tool in biological, pharmaceutical, and clinical studies.

Peanut ( Arachis hypogaea) lectin recognizes α-linked galactose, but not N-acetyl lactosamine in N-linked oligosaccharide terminals

Peanut ( Arachis hypogaea) agglutinin (PNA) is extensively used as tumour marker as it strongly recognises the cancer specific T antigen (Galβ1→3GalNAc-), but not its sialylated version. However, an additional specificity towards Galβ1→4GlcNAc (LacNAc), which is not tumour specific, had been attributed to PNA. For correct interpretation of lectin histochemical results we examined PNA sugar specificity using naturally occurring or semi-synthetic glycoproteins, matrix-immobilised galactosides and lectin-binding tissue glycoproteins, rather than mono- or disaccharides as ligands. Dot-blots, transfer blots or polystyrene plate coatings of the soluble glycoconjugates were probed with horse-radish peroxidase (HRP) conjugates of PNA and other lectins of known specificity. Modifications of PNA-binding glycoproteins, including selective removal of O-linked oligosaccharides and treatment with glycosidases revealed that Galβ1→4GlcNAc (LacNAc) was ineffective while terminal α-linked galactose (TAG) as well as exposed T antigen (Galβ1→3 GalNAc-) was excellent as sugar moiety in glycoproteins for their recognition by PNA. When immobilised, melibiose was superior to lactose in PNA binding. Results were confirmed using TAG-specific human serum anti-α-galactoside antibody.

Jacalin interacts with Asn-linked glycopeptides containing multi-antennary oligosaccharide structure with terminal α-linked galactose

The carbohydrate binding properties of jacalin lectin were examined using RAF9 cell-derived d-[6- 3H]glucosamine-radiolabeled total glycopeptides containing N-linked and O-linked oligosaccharides. The binding of N-linked glycopeptides to jacalin was abolished by treatment of α-galactosidase whereas O-linked glycopeptides were still bound lectin after this treatment. The removal of O-linked oligosaccharides by mild alkaline/borohydride treatment completely eliminated the lectin binding of α-galactosidase treated glycopeptides. These results demonstrate that jacalin interacts with cellular glycopeptides containing N-linked oligosaccharides with terminal α-galactose residues as well as glycopeptides containing O-linked oligosaccharides.

Characterization of Monoclonal Antibody Glycosylation: Comparison of Expression Systems and Identification of Terminal α-Linked Galactose

CAMPATH-1H is a recombinant humanized murine monoclonal immunoglobulin (IgG1) which recognizes the CDw52 antigen of human lymphocytes, and has been the subject of clinical trials for the treatment of non-Hodgkin's lymphoma and rheumatoid arthritis. Peptide mapping by liquid chromatography–mass spectrometry was used to confirm the predicted amino acid sequences and profile glycosylation for two CAMPATH isotypes expressed in a murine myeloma cell line (NS0) and a single isotype expressed in both Chinese hamster ovary (CHO) and NS0 lines. The three major glycoforms identified in CAMPATH are fucosylated biantennary structures, containing zero, one, or two galactose residues. Glycosylation of the IgG1form of CAMPATH expressed in CHO cells is consistent with normal human IgG. However, IgG1and IgG4expressed in NS0 cells include two potentially immunogenic glycoforms which contain either one or two nonreducing terminal α-linked galactose residues. Oligosaccharide structures were characterized by a combination of tandem mass spectrometry, methylation analysis, and exoglycosidase digestion. The strategy used here is designed to be widely applicable to recombinant glycoproteins.

Anomer specificity of the 14 kDa galactose-binding lectin: A reappraisal

A β-anomer preference among galactosides has been attributed to the S-type 14 kDa galactose binding lectin. Here the anomeric preference of this lectin from bovine brain (BBL) is reexamined using inhibition of lectin-mediated haemagglutination, binding of the lectin to dot-blotted glycoproteins and affinity electrophoresis of the lectin through polysaccharide-containing gels. 1.0-methyl α-D-galactoside was 8 times better inhibitor of BBL than the corresponding s-anomer. The terminal galactose in bovine thyroglobulin (exclusively. α-linked) were also nearly 8 times more inhibitory than those in asialofetuin (exclusively s-linked). The terminal α-galactose-containing endogenous glycoproteins of bovine brain were nearly 4 times better inhibitors of BBL than laminin. Removal of terminal α-galactose units by α-galactosidase fully abolished the BBL binding of thyroglobulin and endogenous glycoproteins. BBL was also sugar-specifically retarded by polyacrylamide gel containing guar galactommannan which bears only α-linked galactose. Data indicated that α-galactosides were sometimes better than their β-anomers in binding to BBL. The significance of this observation to the physiological role of galactose-binding lectins is discussed.

Human α-galactosidase gene expression: significance of two peptide regions encoded by exons 1–2 and 6

Two proteins with α-galactosidase activity, α-galactosidase A (α-GalA) and α-galactosidase B (α-GalB, or α- N-acetylgalactosaminidase; α-NAGA) have a high homology of amino-acid sequence. Point mutations of the α-GalA gene have been reported only in the exons 1, 2 or 6. In this study, the exon 1–2 and/or 6 sequences of α-GalA cDNA were partly substituted by the corresponding regions of α-GalB cDNA, and three chimeric proteins were prepared by the baculovirus expression system: CMB12 with substitution at the exon 1–2 region, CMB6 at the exon 6 region, and CMB126 at both regions. They all preserved α-GalA antigenicity. Their kinetic properties toward 4-methylumbelliferyl α-galactopyranoside were compared with those of α-GalA. The catalytic activity was slightly low in CMB12, decreased to 1 2 in CMB6, and restored to a significant degree in CMB126. K m was more than 4-fold higher for CMB6 and CMB126 than for α-GalA. The pH optimum was 4.0 for both CMB12 and α-GalA, 4.8 for CMB6, and 4.6 for CMB126 and α-GalB. The catalytic activity was inhibited most by galactosamine in CMB6, and less in α-GalB, CMB126, α-GalA and CMB12 in decreasing order. The 50% inhibition concentrations of melibiose (Galα1-6Glc) and methyl α-galactopyranoside were 2.5- to 3-fold higher for CMB126 than for α-GalA. These results indicate that the low affinity of CMB126 to the substrate was caused by a reduced affinity to terminal α-linked galactose. We conclude that (1) the two regions encoded by exons 1–2 and 6 contribute to the α-galactosidic cleavage, and (2) an increase in K m of CMB6 or CMB126, with chimeric substitutions at the exon 6 region, was caused by a loss of affinity toward terminal α-linked galactose.