Naohisa Kochibe

Growth retardation and early death of beta-1,4-galactosyltransferase knockout mice with augmented proliferation and abnormal differentiation of epithelial cells.

Carbohydrate chains on a glycoprotein are important not only for protein conformation, transport and stability, but also for cell–cell and cell–matrix interactions. UDP‐Gal:N‐acetylglucosamine β‐1,4‐galactosyltransferase (GalT) (EC 2.4.1.38) is the enzyme which transfers galactose (Gal) to the terminal N‐acetylglucosamine (GlcNAc) of complex‐type N‐glycans in the Golgi apparatus. In addition, it has also been suggested that this enzyme is involved directly in cell–cell interactions during fertilization and early embryogenesis through a subpopulation of this enzyme distributed on the cell surface. In this study, GalT‐deficient mice were produced by gene targeting in order to examine the pathological effects of the deficiency. GalT‐deficient mice were born normally and were fertile, but they exhibited growth retardation and semi‐lethality. Epithelial cell proliferation of the skin and small intestine was enhanced, and cell differentiation in intestinal villi was abnormal. These observations suggest that GalT plays critical roles in the regulation of proliferation and differentiation of epithelial cells after birth, although this enzyme is dispensable during embryonic development.

Systematic fractionation of oligosaccharides of human immunoglobulin G by serial affinity chromatography on immobilized lectin columns

Human immunoglobulin G is known to contain 16 different biantennary complex-type asparagine-linked sugar chains, each of which occurs in a nonsialylated, monosialylated, or disialylated form. These oligosaccharides can be separated into 14 fractions by sequential affinity chromatography with Aleuria aurantia lectin (AAL)-Sepharose, RCA120-WG003, and E4-phytohemagglutinin-agarose columns. Twelve of them were found to contain a single oligosaccharide, while the fraction which passed through all three columns was shown to contain two oligosaccharides, GlcNAc beta 1----2Man alpha 1----6(+/- GlcNAc beta 1----4) (GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcOT. The fraction, which bound to the AAL-Sepharose column and passed through the remaining two lectin columns, also contained two oligosaccharides, GlcNAc beta 1----2Man alpha 1----6(+/- GlcNAc beta 1----4) (GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4 (Fuc alpha 1----6)GlcNAcOT. These results indicated that serial affinity chromatography with the three lectin columns can be used effectively to detect changes in the sugar chains of IgG resulting from diseases such as rheumatoid arthritis.

α1‐Acid glycoprotein fucosylation as a marker of carcinoma progression and prognosis

Serum α1‐acid glycoprotein (AGP), an acute‐phase protein secreted by the liver, carries α(1,3)‐fucosylated structures on its 5 highly branched, N‐linked sugar chains.

Structural study of the sugar moieties of monoclonal antibodies secreted by human-mouse hybridoma☆

Six monoclonal antibodies, three each of human IgG1 and IgG2 subclasses, were obtained from human-mouse hybridomas. Structural study of their asparagine-linked sugar chains was performed to elucidate the regulatory mechanism of secreted monoclonal IgG glycosylation. The sugar moieties were quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. They were converted into radioactive oligosaccharides by NaB3H4 reduction after N-acetylation. Structural study of each oligosaccharide by lectin affinity column chromatography, sequential exoglycosidase digestion, and methylation analysis indicated that almost all of them were biantennary complex-type sugar chains containing Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4 (+/- Fuc alpha 1----6)GlcNAc as core structures. Bisecting N-acetylglucosamine residue, which is present in human IgG but not in mouse IgG, could not be detected at all. The molar ratio of each oligosaccharide from the six IgG samples was different. However, no subclass specificity was detected except that all IgG1 contained neutral, mono-, and disialylated sugar chains, whereas IgG2 did not contain disialylated ones. The molar ratio of N-acetylneuraminic acid to N-glycolylneuraminic acid was also different for each IgG. All six IgGs contained monoantennary complex-type and high mannose-type oligosaccharides which had never been detected in serum IgGs of various mammals so far investigated. These results indicated that the processing of asparagine-linked sugar chains of IgG is less complete in human-mouse hybridoma than in human or mouse B cells, and that the glycosylation machinery of the mouse cells is dominant in the hybrid cells.

Cloning and expression of a functional fucose-specific lectin from an orange peel mushroom, Aleuria aurantia

Aleuria aurantia lectin (AAL) shows sugar‐binding specificity for L‐fucose. A λgt11 expression library was constructed from A. aurantia poly(A) RNA and screened with a polyclonal antiserum directed against AAL. An immunopositive clone carrying 1.3‐kb EcoRI fragment was obtained. The fragment encoded AAL, but lacked a nucleotide sequence corresponding to the two amino‐terminal amino acids. The 5′‐terminal part of the fragment was replaced with a chemically synthesized DNA fragment and inserted into an expression vector to yield a plasmid pKA‐1. Escherichia coli carrying pKA‐1 expressed functional AAL and the recombinant AAL showed the same immunological properties as those of natural AAL.

Crystallization and characterization of a lectin obtained from a mushroom, Aleuria aurantia

A fucose-specific lectin with a unique sugar recognizing property was purified from an orange peel mushroom, Aleuria aurantia, by using a specific affinity adsorbent prepared from L-fucose and starch. From 100 g of fruiting bodies, 145 mg of pure lectin was obtained. The lectin was crystallized and the crystals showed hexagonal bipyramid in shape. Distribution of hydrophobic and hydrophilic regions in the molecule of this lectin was predicted from the amino acid sequence deduced from the previously reported nucleotide sequence of the lectin cDNA. Circular dichroism spectra revealed a very low content of alpha-helical and beta-sheet structures and a relatively high content of turns in this lectin. From the spectrum observed in the presence of L-fucose, a hapten sugar of this lectin, certain conformational change was assumed to occur.

Structural study of the carbohydrate moieties of two human immunoglobulin subclasses (IgG2 and IgG4)

Asparagine-linked sugar chains were quantitatively released as oligosaccharides from human IgG2 and IgG4 myeloma proteins by hydrazinolysis followed byN-acetylation and NaB3H4 reduction. Each oligosaccharide was isolated by serial lectin column chromatography. Study of their structures by sequential exoglycosidase digestion and methylation analysis, revealed that all of them were of the bi-antennary complex-type containing Manα1-6(±GlcNacß1-4)(Manα1-3)Manß1-4GlcNAcß1-4(±Fucα1-6)GlcNAc, as core structures, and GlcNAcß1-, Galß1-4GlcNacß1- and Siaα2-6Galß1- in their outer chain moieties. However, the molar ratio of each oligosaccharide was different in each IgG sample, indicating that clonal variation is included in the sugar chain moieties of IgG molecules. One of the IgG2 contained four asparagine-linked sugar chains in one molecule, two on the Fc fragment and the remainder on the Fab fragment. The sugar chains in the Fc fragment contained much less galactose as compared with the Fab fragment.

Yearly and Seasonal Changes of Specific IgE to Japanese Cedar Pollen in a Young Population

BACKGROUND There have been no detailed long-term observations of the relationship between specific IgE production and stimulation by various naturally occurring allergens. OBJECTIVE This study was conducted to elucidate the yearly and seasonal changes of specific IgE antibody production to Japanese cedar pollen, an allergen of Japanese cedar pollinosis, in young adults. METHODS The number of Japanese cedar pollen were counted over a period of 9 years. Changes in the percentages of antibody carriers to Japanese cedar pollen and mite were examined during these years. Changes in Japanese cedar pollen-specific IgE levels between a low exposure year and a high exposure year in individual subjects were also investigated. RESULTS From 1987 to 1995, the percentages of Japanese cedar pollen-IgE carriers varied from about 30% to 50% with the intensity of pollen stimulation, and carriers tended to increase yearly. The rates of anti-mite IgE carriers changed little. In the spring which is the pollen season, Japanese cedar pollen-IgE levels in low exposure years were weaker than those in high exposure years in individual subjects. Levels in autumn, which is not the pollen season, showed equivalent levels in both high and low exposure years. Anti-mite IgE levels in individual subjects varied little during these years. CONCLUSIONS A long-term follow-up study supported that Japanese cedar pollen-IgE production is mainly associated with the degree of allergen exposure.

Structural studies of the asparagine-linked sugar chains of two immunoglobulin M's purified from a patient with Waldenström's macroglobulinemia☆

The structures of the sugar chains present in two human monoclonal IgM molecules purified from the serum of a patient with Waldenströms macroglobulinemia have been determined. The asparagine-linked sugar chains were liberated as oligosaccharides by hydrazinolysis and labeled by reduction with NaB3H4 after N-acetylation. Their structures were studied by serial lectin column chromatography and sequential exoglycosidase digestion in combination with methylation analysis. These two IgMs were shown to contain almost the same sugar chains. The sugar chains were a mixture of a series of high-mannose-type and biantennary complex-type oligosaccharides. The complex-type oligosaccharides contain Man alpha 1----6(+/- GlcNAc beta 1----4)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----6)GlcNAc as their core and GlcNAc beta 1----, Gal beta 1----4GlcNAc beta 1---- and Neu5Ac alpha 2----6Gal beta 1----4GlcNAc beta 1---- groups in their outer chain moieties.

Structural differences found in the sugar chains of eutopic and ectopic free α-subunits of human glycoprotein hormone

Free alpha-subunits of human glycoprotein hormone were purified from the urine of a healthy pregnant woman and from that of a patient with adenocarcinoma. Comparative study of their sugar moieties revealed that they have different numbers and different sets of asparagine-linked sugar chains, which are also different from those of alpha-subunit obtained by dissociation of whole hCG molecule. The eutopic free alpha-subunit contained biantennary complex-type sugar chains only. In contrast, the ectopic free alpha-subunit contained tri- and tetraantennary complex-type sugar chains in addition.