Α-galactosyl Residues Research Articles

Lectin histochemistry reveals two cytotrophoblast differentiation pathways during placental development in the feline (Feliscatus)

IntroductionPlacental glycosylation has been examined on eight feline placentae ranging from approximately 15 to 60 days post-conception as little is known about changes in glycan distribution in this species. MethodsSpecimens were resin embedded and lectin histochemistry was applied to semi-thin sections using a panel of 24 lectins and an avidin-biotin revealing system. ResultsAbundant tri-tetraantennary complex N-glycan and α-galactosyl residues found in the syncytium in early pregnancy were greatly reduced in mid-pregnancy, though retained at the invasion front in the syncytium (N-glycan) or cytotrophoblast layer (αGal). Some other glycans were also uniquely present in invading cells. Abundant polylactosamine was found in the infolding basal lamina of syncytiotrophoblast and the apical villous cytotrophoblast membrane. Syncytial secretory granules often clustered near the apical membrane abutting maternal vessels. Decidual cells selectively expressed β-galactosyl residues throughout pregnancy and highly branched N-glycan levels increased over time. DiscussionGlycan distribution changes significantly over pregnancy, probably relating to the development of transport and invasive properties of trophoblast which in the endotheliochorial placenta reaches the level of the maternal vessels. Highly branched complex N-glycans, often associated with invasive cells, N-Acetylgalactosamine and terminal α-galactosyl residues are present at the invasion front abutting the junctional zone of the endometrium. Abundant polylactosamine on the syncytiotrophoblast basal lamina may reflect the presence of specialised adhesive interactions, while clustering of glycosylated granules apically is probably associated with secretion and absorption of material via maternal vasculature. It is suggested that lamellar and invasive cytotrophoblast represent distinct differentiation pathways. 246 words.

Specificity profile of αGal antibodies in αGalT KO mice as probed with comprehensive printed glycan array: Comparison with human anti-Galili antibodies.

The α1,3-galactosyltransferase gene-knockout (GalT KO) mice are able to produce natural anti-αGal antibodies apparently without any specific immunization. GalT KO mice are commonly used as a model immunological system for studying anti-αGal responses to Gal-positive xenografts in human. In this study, we compared the specificity of mouse and human αGal antibodies to realize the adequacy of the murine model. Using hapten-specific affinity chromatography antibodies against Galα1-3Galβ1-4GlcNAcβ epitope were isolated from both human and GalT KO mice blood sera. Specificity of isolated antibodies was determined using a printed glycan array (PGA) containing 400 mammalian glycans and 200 bacterial polysaccharides. The quantity of isolated specific anti-Galα antibodies corresponds to a content of <0.2% of total Ig, which is an order of magnitude lower than that generally assumed for both human and murine peripheral blood immunoglobulin, with a high predominance of IgM over IgG (95% vs 5%). Analysis using a printed glycan array has demonstrated that (a) antibodies from both species bind not only the Galα1-3Galβ1-4GlcNAcβ epitope, but also unrelated glycans; (b) particularly, for human (but not mouse) antibodies the best binders appear to be bacterial polysaccharides; (c) the profile of mouse antibodies is broader, it is noteworthy that they recognize a variety of human blood group B epitopes and even glycans without the α-galactosyl residue. We believe that the mouse model should be used cautiously in xenotransplantation experiments when the fine epitope specificity of antibodies is critical.

A novel α-galactosidase from Fusarium oxysporum and its application in determining the structure of the gum arabic side chain.

We previously reported that Fusarium oxysporum 12S produces two bifunctional proteins, FoAP1 and FoAP2, with α-d-galactopyranosidase (GPase) and β-l-arabinopyranosidase (APase) activities. The aim of this paper was to purify a third GPase, FoGP1, from culture supernatant of F. oxysporum 12S, to characterize it, and to determine its mode of action towards gum arabic. A cDNA encoding FoGP1 was cloned and the protein was overexpressed in Escherichia coli. Module sequence analysis revealed the presence of a GH27 domain in FoGP1. The recombinant enzyme (rFoGP1) showed a GPase/APase activity ratio of 330, which was quite different from that of FoAP1 (1.7) and FoAP2 (0.2). Among the natural substrates tested, rFoGP1 showed the highest activity towards gum arabic. In contrast to other well-characterized GPases, rFoGP1 released a small amount of galactose from α-galactosyl oligosaccharides such as raffinose and exhibited no activity toward galactomannans, which are highly substituted with α-galactosyl side chains. This indicated that FoGP1 is an unusual type of GPase. rFoGP1 released 30% of the total galactose from gum arabic, suggesting the existence of a large number of α-galactosyl residues at the non-reducing ends of gum arabic side chains. Together, rFoGP1 and α-l-arabinofuranosidase released four times more arabinose than α-l-arabinofuranosidase acting alone. This suggested that a large number of α-l-arabinofuranosyl residues is capped by α-galactosyl residues. 1H NMR experiments revealed that rFoGP1 hydrolyzed the α-1,3-galactosidic linkage within the side chain structure of [α-d-Galp-(1→3)-α-l-Araf-(1→] in gum arabic. In conclusion, rFoGP1 is highly active toward α-1,3-galactosyl linkages but negligibly or not active toward α-1,6-galactosyl linkages. The novel FoGP1 might be used to modify the physical properties of gum arabic, which is an industrially important polysaccharide used as an emulsion stabilizer and coating agent.

Multiplex Analysis of Novel Urinary Lyso-Gb3-Related Biomarkers for Fabry Disease by Tandem Mass Spectrometry

Fabry disease is a lysosomal storage disorder caused by the absence or reduction of α-galactosidase A enzyme activity. The enzymatic deficiency results in the impaired catabolism of neutral sphingolipids with terminal α-galactosyl residues and subsequent accumulation in several tissues. Biomarkers reflecting disease severity and progression, the response to therapeutic intervention, and details of molecular pathogenesis are needed. Until now, two sphingolipids were targeted as biomarkers in urine and plasma of Fabry patients: globotriaosylceramide (Gb(3)) and globotriaosylsphingosine (lyso-Gb(3)). Using metabolomic approaches, our group recently discovered seven novel urinary lyso-Gb(3)-related Fabry disease biomarkers with mass-to-charge ratios (m/z) of 758, 774, 784, 800, 802, 820, and 836. All these biomarkers exhibited modifications of the lyso-Gb(3) sphingosine moiety. The aims of the present study were to devise and validate a specific tandem mass spectrometry multiplex methodology for the relative quantification of these seven analogues and to evaluate their urinary excretion levels in samples from 164 Fabry patients and 94 healthy controls. We found no detectable analogues in healthy controls, except for trace amounts of the analogue with m/z 836. Significant correlations were established between lyso-Gb(3) analogue levels in urine and gender (p < 0.001). Fabry males had higher excretion levels compared to females with the disease. Lyso-Gb(3) analogue levels correlated well with enzyme replacement therapy (ERT) status in males (p < 0.05). The urinary analogue distributions varied among Fabry patients. However, the analogues with m/z 802, 820, and 836 were generally more abundant in the majority of patients. Lyso-Gb(3) analogues are promising urinary biomarkers for Fabry disease.

Intraspecies Variation in Trypanosoma cruzi GPI-Mucins: Biological Activities and Differential Expression of α-Galactosyl Residues

The glycosylphosphatidylinositol (GPI)-anchored mucins of Trypanosoma cruzi trypomastigotes play an important immunomodulatory role during the course of Chagas disease. Here, some biological activities of tGPI-mucins from four T. cruzi isolates, including benznidazole-susceptible (BZS-Y), benznidazole-resistant (BZR-Y), CL, and Colombiana, were evaluated. GPI-mucins were able to differentially trigger the production of interleukin-12 and nitric oxide in BALB/c macrophages and modulate LLC-MK2 cell invasion. The significance of these variations was assessed after analysis of the terminal α-galactosyl residues. Enzymatic treatment with α-galactosidase indicated a differential expression of O-linked α-galactosyl residues among the strains, with higher expression of this sugar in BZS-Y and BZR-Y T. cruzi populations followed by Colombiana and CL. Unweighted pair group method analysis of the carbohydrate anchor profile and biological parameters allowed the clustering of two groups. One group includes Y and CL strains (T. cruzi II and VI), and the other group is represented by Colombiana strain (T. cruzi I).

Terminal galactosylation of glycoconjugates in Plasmodium falciparum asexual blood stages and Trypanosoma brucei bloodstream trypomastigotes

There is definitive biochemical evidence for the presence of terminal α-galactosyl residues (α-gal) in the N-linked oligosaccharides and glycophosphatidylinositol anchors (GPI anchors) of the variant surface glycoprotein of Trypanosoma brucei bloodstream trypomastigotes. Indirect evidence also exists for α-gal in Plasmodium falciparum asexual blood stage glycoproteins and glycolipids. The occurrence of α-gal in glycoproteins and glycolipids of T. brucei bloodstream trypomastigotes and P. falciparum late asexual blood stages was investigated by the binding of α-gal-specific Bandeirea simplicifolia B4 lectin 1 (BSB4), incorporation of [3H]galactose from UDP-[3H]galactose into glycoproteins and glycolipids in microsomes in vitro, and bioinformatic searches for galactosyl-transferase coding sequences. The findings confirm the presence of α-gal in a spectrum of T. brucei bloodstream trypomastigote glycoproteins and glycolipids and indicate its relative absence from P. falciparum asexual blood stage glycoconjugates.

Classification, Substrate Specificity and Application of α-Galactosidases

Galactose is found in many oligosaccharides, galactomannans, glycoproteins and glycolipids, which are widely distributed in plants microorganisms and animals. α-Galactosidase (α-Gal) catalyzes the hydrolysis of 1,6-linked α-galactosyl residues and transgalactosylation. α-Gals are classified into four glycoside hydrolases families (GH): 4, 27, 36 and 57. The majority of known α-Gals belongs to GH families 27 and 36.α-Gals are of particular interest in view of their biotechnological applications.

The proteome of Trypanosoma cruzi shed vesicles involved in host immunomodulation and cell invasion

Infective trypomastigote forms of Trypanosoma cruzi release vesicles to the culture medium that are rich in α-galactosyl residues (TcαGalVes). These vesicles induce potent proinflammatory host immune response and greatly enhance host cell invasion by the parasite. For proteomic analysis, the conditioned medium from cell-derived trypomastigotes was fractionated by gel-filtration, followed by affinity chromatography using anti-αGal antibodies. TcαGalVes were digested with proteases using three different strategies. To increase protein coverage, released peptides were fractionated by strong cation-exchange chromatography followed by reverse-phase chromatography, and analyzed by ESI-MS/MS. MS/MS spectra were correlated to TcruziDB v3.0 using Sequest, Phenyx and Mascot algorithms. We identified 126 proteins, including several members of the trans-sialidase (TS)/gp85 and gp63 superfamilies, known to play a key role in host cell adhesion and invasion by the parasite. We also found several polypeptides related to mammalian cell exosomes. Our data clearly show that TcαGalVes contain the major virulence factors of T. cruzi, responsible for promoting the parasite entry into the host cell, and for its evasion from the host immune response. Supported by BBRC/Biology (NIH#5G12RR008124), FAPESP, and the Wellcome Trust.

Two Novel Types of O-Glycans on the Mugwort Pollen Allergen Art v 1 and Their Role in Antibody Binding

Art v 1, the major allergen of mugwort (Artemisia vulgaris) pollen contains galactose and arabinose. As the sera of some allergic patients react with natural but not with recombinant Art v 1 produced in bacteria, the glycosylation of Art v 1 may play a role in IgE binding and human allergic reactions. Chemical and enzymatic degradation, mass spectrometry, and 800 MHz (1)H and (13)C nuclear magnetic resonance spectroscopy indicated the proline-rich domain to be glycosylated in two ways. We found a large hydroxyproline-linked arabinogalactan composed of a short beta1,6-galactan core, which is substituted by a variable number (5-28) of alpha-arabinofuranose residues, which form branched side chains with 5-, 2,5-, 3,5-, and 2,3,5-substituted arabinoses. Thus, the design of the Art v 1 polysaccharide differs from that of the well known type II arabinogalactans, and we suggest it be named type III arabinogalactan. The other type of glycosylation was formed by single (but adjacent) beta-arabinofuranoses linked to hydroxyproline. In contrast to the arabinosylation of Ser-Hyp(4) motifs in other hydroxyproline-rich glycoproteins, such as extensins or solanaceous lectins, no oligo-arabinosides were found in Art v 1. Art v 1 and parts thereof produced by alkaline degradation, chemical deglycosylation, proteolytic degradation, and/or digestion with alpha-arabinofuranosidase were used in enzyme-linked immunosorbent assay and immunoblot experiments with rabbit serum and with the sera of patients. Although we could not observe antibody binding by the polysaccharide, the single hydroxyproline-linked beta-arabinose residues appeared to react with the antibodies. Mono-beta-arabinosylated hydroxyproline residues thus constitute a new, potentially cross-reactive, carbohydrate determinant in plant proteins.

Fabry disease in childhood

Fabry disease, also known as Anderson-Fabry disease or angiokeratoma corporis diffusum universale, is an inborn error of metabolism with profound clinical consequences. Patients with Fabry disease have a deficiency of α-galactosidase A (α-Gal A), the lysosomal enzyme responsible for the breakdown of globotriaosylceramide and related glycosphingolipids. 1. Brady R.O Gal A.E Bradley R.M Martensson E Warshaw A.L Laster L Enzymatic defect in Fabry's disease: ceramidetrihexosidase deficiency. N Engl J Med. 1967; 276: 1163-1167 Crossref PubMed Scopus (819) Google Scholar These glycosphingolipids have a terminal α-galactosyl residue and are components of most cell membranes. The deficiency of α-Gal A leads to progressive accumulation of globotriaosylceramide in the plasma and in the lysosomes of most cells in the body. The kidney in Fabry diseaseThe Journal of PediatricsVol. 146Issue 1PreviewTo the Editor: Full-Text PDF

Immunoaffinity removal of xenoreactive antibodies using modified dialysis or microfiltration membranes.

Hyperacute rejection following xenogeneic transplantation in primates is mediated by naturally occurring IgM antibodies, which are specifically directed to alpha-Galactosyl residues on many nonprimate mammalian cells. Current approaches to remove these anti-alphaGal IgM include plasmapheresis followed by immunoaffinity adsorption on bead columns using synthetic Gal epitopes, which requires two pieces of complex equipment. In this study, we explored the use of immunoaffinity adsorption with hollow fiber microporous or dialysis membranes to which a synthetic alphaGal trisaccharide ligand is bound. Covalent attachment of ligand directly to the surface produced negligible binding, but use of long-chain polyamines as reactive spacers yielded binding densities for anti-alphaGal IgM as high as 89 mg/mL membrane volume in breakthrough curve experiments with microporous nylon membranes having an internal surface area of 4.2 m(2)/mL membrane volume. A crossflow microfilter fabricated from the membranes described in this study and having about 0.4 m(2) luminal surface area would be able to carry out plasma separation and immunoadsorption in a single device with a large excess of binding capacity to ensure that all plasma that filters across the device and is returned to a human patient is essentially free of anti-alphaGal IgM. We conclude that immunoaffinity removal of xenoreactive antibodies using microfiltration hollow fiber membranes is feasible and has potential advantages of efficiency and simplicity for clinical application.

α-Galactosidase from cultured rice ( Oryza sativa L. var. Nipponbare) cells

The α-galactosidase from rice cell suspension cultures was purified to homogeneity by different techniques including affinity chromatography using N-ϵ-aminocaproyl-α- d-galactopyranosylamine as the ligand. From 11 l of culture filtrate, 28.7 mg of purified enzyme was obtained with an overall yield of 51.9%. The cDNA coding for the α-galactosidase was cloned and sequenced. The enzyme was found to contain 417 amino acid residues composed of a 55 amino acid signal sequence and 362 amino acid mature α-galactosidase; the molecular weight of the mature enzyme was thus calculated to be 39,950. Seven cysteine residues were also found but no putative N-glycosylation sites were present. The observed homology between the deduced amino acid sequences of the mature enzyme and α-galactosidases from coffee ( Coffea arabica), guar ( Cyamopsis tetragonolooba), and Mortierella vinacea α-galactosidase II were over 73, 72, and 45%, respectively. The enzyme displayed maximum activity at 45 °C when p-nitrophenyl-α- d-galactopyranoside was used as substrate. The rice α-galactosidase and Mortierella vinacea α-galactosidase II acted on both the terminal α-galactosyl residue and the side-chain α-galactosyl residue of the galactomanno-oligosaccharides.

Renal Pathology in Fabry Disease

Fabry disease is an X-linked recessive lysosomal storage disease that is caused by deficient activity of the lysosomal enzyme α-galactosidase A (α-Gal A), an enzyme that cleaves terminal α-galactosyl residues. This deficiency results in progressive lysosomal accumulation of glycosphingolipid with

High Overexpression of the Human α-Galactosidase A Gene Driven by Its Promoter in Transgenic Mice: Implications for the Treatment of Fabry Disease

BackgroundHuman α-galactosidase A (α-Gal A) is the lysosomal enzyme that cleaves α-galactosyl residues from glycoconjugates and is the deficient enzyme in Fabry disease. To date, there have been no studies...

Structural Analysis of the Asparagine-Linked Glycan Units of the ZP2 and ZP3 Glycoproteins from Mouse Zona Pellucida

Zona pellucida (ZP), the extracellular glycocalyx that surrounds the mammalian egg plasma membrane, is a relatively simple structure consisting of three to four glycoproteins. In the mouse, the ZP is composed of three glycoproteins, namely ZP1 (200 kDa), ZP2 (120 kDa), and ZP3 (83 kDa). Extensive studies in this species have resulted in the identification of primary (mZP3) and secondary (mZP2) binding sites for spermatozoa. The two zona components are highly glycosylated containing N-linked and O-linked glycan units. In an attempt to characterize N-linked glycan units, mZP2 and mZP3 were purified and the N-linked carbohydrate chains were released by exhaustive digestion with N-glycanase. The released oligosaccharides (OSs) were radiolabeled by reduction with NaB3H4 and resolved by gel filtration on a column of Bio-Gel P-4. The OSs separated into several peaks indicating the presence of a variety of N-linked glycans. Interestingly, the radioactive peaks resolved from mZP2 and mZP3 were quite different, a result suggesting qualitative and quantitative differences in the glycans. The [3H]-labeled glycans present in mZP2 and mZP3 were pooled separately and fractionated by serial lectin chromatography. Experimental evidence included in this report strongly suggests that mZP3 (but not mZP2) contains polylactosaminyl glycan with terminal, nonreducing α-galactosyl residues. The mZP3 glycans eluted from the immobilized lectin columns were further characterized by lectin and sizing column chromatography before or after digestion with endo-/exo-glycohydrolases. Data revealed the presence of a variety of OSs, including poly-N-acetyllactosaminyl, bi-, tri-, and tetraantennary complex-type, and high-mannose-type glycans. Taken together, these results provide additional evidence on the complex nature of the glycan chains present on mZP glycoconjugates.

Removal of terminal α-Galactosyl residues by endo-β-galactosidase C, a potent endoglycosidase which acts under physiological pH conditions

Fabry disease is an X-linked recessive inborn error of glycosphingolipid catabolism caused by the deficient activity of the lysosomal enzyme, α-galactosidase A (α-Gal A). This enzymatic defect results in the progressive accumulation of globotriaosylceramide (GL-3) and related glycosphingolipids with terminal α-galactosyl moieties in the lysosomes of endothelial, epithelial, perithelial, and smooth muscle cells throughout the body. In classically affected males, who have essentially no α-Gal A activity, clinical manifestations result predominantly from the progressive lysosomal deposition of GL-3 in the vascular endothelium. Onset usually occurs in childhood or adolescence with periodic crises of severe pain in the extremities (acroparesthesias), the appearance of vascular cutaneous lesions (angiokeratomas), hypohidrosis, and the characteristic corneal and lenticular opacities. Episodic crises of agonizing, burning pain in the distal extremities most often begin in childhood or early adolescence and signal clinical onset of the disease. These crises typically last from minutes to several days and usually are triggered by exercise, fatigue, emotional stress, or rapid changes in temperature and humidity. The early acroparesthesias that occur in children presumably result from glycosphingolipid deposition in the microvasculature that supplies blood to the peripheral nerve cells. The endothelial glycosphingolipid accumulation narrows the vascular lumen and vessel spasms or frank infarction cause the excruciating pain. Angiokeratomas appear as clusters of individual punctate, dark red to blue-black angiectases in the superficial layers of the skin, and are often one of the earliest manifestations of the disease. Most classically affected males develop cardiovascular and/or cerebrovascular disease in middle age. The progressive vascular involvement is a major cause of morbidity and mortality, particularly after treatment of the renal insufficiency by chronic dialysis or transplantation.

Modification at C6 of the Terminal Galactosyl Residues of Cobra Venom Factor Abolishes Anti-α-Gal Antibody Immunoreactivity without Affecting Functional Activity

TheN-linked oligosaccharides of cobra venom factor (CVF) contain unique terminal α-galactosylated Lewis X structures. We have previously shown that CVF immobilized on nylon membranes binds naturally occurring human anti-α-Gal antibody. The present study shows that soluble CVF can effectively inhibit the binding of anti-α-Gal antibody to CVF-coated microtiter plates, indicating that the terminal α-galactosyl residues of the functionally active CVF are accessible to anti-α-Gal antibody binding. Modification of the terminal galactosyl residues of CVF by treatment with galactose oxidase andin situderivatization of the generated aldehyde groups with hydrazides abolished the human anti-α-Gal antibody immunoreactivity without affecting the complement-activating activity.

Alpha-Galactosidase A deficient mice: a model of Fabry disease.

Fabry disease is an X-linked inherited metabolic disorder that is caused by a deficiency of alpha-galactosidase A (alpha-Gal A). Progressive deposition of neutral glycosphingolipids that have terminal a-linked galactosyl moieties in vascular endothelial cells causes renal failure along with premature myocardial infarctions and strokes in patients with this condition. No specific treatment is available for patients with this disorder at this time. An animal model of this condition would be valuable for exploring therapeutic strategies for patients with Fabry disease. We report here the generation of alpha-Gal A deficient mice by gene targeting and an analysis of the resulting phenotype. The knockout mice display a complete lack of alpha-Gal A activity. The mice, however, appeared clinically normal at 10 weeks of age. Ultrastructural analysis revealed concentric lamellar inclusions in the kidneys, and confocal microscopy using a fluorescent-labeled lectin specific for alpha-D-galactosyl residues showed accumulation of substrate in the kidneys as well as in cultured fibroblasts. Lipid analysis revealed a marked accumulation of ceramidetrihexoside in the liver and the kidneys. These findings indicate the similarity of the pathophysiological process in the mutant mice and in patients with Fabry disease. The deficiency of alpha-Gal A activity and the accumulation of material containing terminal alpha-galactosyl residues in cultured embryonic fibroblasts derived from alpha-Gal A(-/0) mice were corrected by transducing these cells with bicistronic multidrug resistance retroviruses containing human alpha-Gal A cDNA.

Antibody Levels against α-Galactosyl Epitopes in Sera of Patients with Squamous Intraepithelial Lesions and Early Invasive Cervical Carcinoma

We measured serum levels of anti-Gal(α1 → 3)Gal and anti-Gal(α1 → 2)Gal antibodies in 89 and 91 women, respectively, by using ELISA. These patients had cervical intraepithelial neoplasia (CIN) grades 1 to 3 and early invasive cervical carcinoma (ICC). Our objective was to compare anti-α-galactosyl antibody levels among them and with those of normal controls. High levels of anti-Gal(α1 → 2)Gal antibodies were detected in 22% of patients (P= 0.006). The mean level was 1.6 times greater than that of controls, without difference among subgroups. Thirty percent of patients had abnormally high anti-Gal levels (P= 0.001). Mean levels were twofold greater than the mean control value. Subsets with human papillomavirus/CIN 1 and CIN 2–3 had high immunoreactivity (P= 0.004). Both antibodies showed a significant correlation (r= 0.53,P< 0.00001). We conclude that 22 to 30% of patients with CIN 1–3 showed significantly high levels of anti-α-galactosyl antibodies. This seroreactivity might be related to the abnormal expression of α-galactosyl residues at some point of the natural history of human papillomavirus infection of the uterine cervix, suggesting an active immune response by natural antibodies against this virus. Further studies are needed to determine whether anti-α-galactosyl antibodies confer protection in human papillomavirus infection.

Tissue Targeting and Plasma Clearance of Cobra Venom Factor in Mice

The tissue targeting and rate of clearance of cobra venom factor (CVF) from the circulation was studied in mice by intravenous or intraperitoneal injection of radioiodinated CVF. In both modes of administrations, CVF was targeted mainly to liver. CVF injected directly into the blood was cleared from the circulation with a plasma half life of about 10 h, whereas CVF injected into the peritoneal cavity was slowly absorbed into the blood stream reaching a maximum level at ∼6 h, and it was then cleared from the circulation with a plasma half life of about 18 h. The rate of plasma clearance of CVF was markedly decreased upon removal of the terminal α-galactosyl residues of the oligosaccharide chains; the plasma half lives for intravenously and intraperitoneally administered de-α-galactosylated CVF were ∼5 and ∼10 h, respectively. However, the clearance rate was not affected by complete deglycosylation usingN-glycanase or by chemical modification of the terminal galactosyl residues. Together, these data demonstrate that the terminal α-galactosyl residues of CVF mask the Lewis X-dependent uptake of CVF by liver.