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 by N-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 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 core structures, and GlcNAc beta 1-, Gal beta 1-4GlcNAc beta 1- and Sia alpha 2-6Gal beta 1- 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.     

Colony structure of a bamboo-dwellingTetraponera sp. (Hymenoptera: Formicidae: Pseudomyrmecinae) from Malaysia

Summary The colony structure of the bamboo-inhabiting SE-Asian pseudomyrmecine antTetraponera sp. PSW-80 nearattenuata F. Smith was investigated at the Ulu Gombak Field Studies Centre, Selangor, West-Malaysia. Based on the dissection of 54 stem internodes from 12 different culms of the large bambooGigantochloa scortechinii Gamble and on the mapping of three colonies, the following demographic characteristics emerge. The colonies are monogynous but highly polydomous (at least up to 36 internodes and up to 9 stems occupied) and very populous for a pseudomyrmecine not involved in an ant-plant mutualism. One completely censused colony had 6953 adult workers and 2079 alates (adults plus pupae). The single queen suppresses gyne development in her own nest and, to a lesser extent, in other nests within the same stem. The overall numerical sex ratio was 0.961 (females:males), the investment sex ratio, 2.931, i.e., almost exactly the 31 ratio expected for a monogynous outbred hymenopteran in which the colony queen also produces all the male offspring. Brood is distributed to all other nest chambers from the queenright chamber. The symbiotic pseudococcids (Kermicus wroughtoni Newstead) are present in all inhabited internodes, with small early instar individuals prevailing numerically by far over the larger stages. The rieht well secluded honeydew supply within the internode and the efficient architectural protection provided by the internode wall (access usually only through a 2 × 3 mm-hole) allowT. sp. PSW-80 to reach an unusually large colony size without being an aggressive and protective plant mutualist like other members of its subfamily with similar demographic features.     

Polymorphism of glycophorins in nonhuman primate erythrocytes

Using immunoblotting techniques and polyclonal antisera to human erythrocyte glycophorin, we show that erythrocytes of several species of nonhuman primates, including representatives of anthropoid apes (19 chimpanzees, 3 gorillas, 6 orangutans, and 3 gibbons) and Old World monkeys (3 baboons, 5 rhesus monkeys, and 6 cynomologus macaques), contain human glycophorin-like molecules. Each species displays a unique glycophorin profile; in anthropoid apes the profile is more complex than in Old World monkeys and more similar to that seen in humans. The chimpanzee was the only species in which human -like glycophorin was detected but it differed from its human counterpart in electrophoretic mobility and reaction with M-specific monoclonal antibody. In contrast to humans, highly polymorphic glycophorin profiles were observed in each species of anthropoid apes and three distinct patterns were defined in each. No such polymorphism has been found so far among the Old World monkeys in the limited number of animals studied. The major glycophorins in all species but the chimpanzees failed to react with M- or N-specific monoclonal antibodies, suggesting structural differences from the human within the amino terminal regions. The reaction with the minor glycophorins showed inter- and intraspecies variability. All glycophorins, except -like glycophorin in the chimpanzee, reacted with the antiserum to the carboxyl terminal fragment of human glycophorin, indicating a structural relation to the human in this region. An unexpected correlation was observed, in the chimpanzee, between the patterns of electrophoretically resolved glycophorins and the V-A-B-D blood-group phenotypes, allowing the assignment of each determinant to specific glycophorin bands. The basis for the differences observed between human and nonhuman primate glycophorins is not clear but the possibilities include a common nonpolymorphic ancestor and differences in selective pressures.This research was supported by National Institutes of Health Grant 5 RO1 GM16389.     

Membrane glycophorins of Dantu blood group erythrocytes

Glycophorins of erythrocytes of two unrelated individuals who exhibit the Dantu blood group phenotype were studied. Immunoblots indicated that erythrocytes of each individual contained a complement of a normal alpha-glycophorin (glycophorin A) and a variant N-glycophorin. delta-Glycophorin (glycophorin B) was present in one donor's cells but not the other's; the s and N phenotypes of the latter's erythrocytes may derive from the variant glycophorin. The variant glycophorin is of a smaller size, does not bind to Lens culinaris lectin agarose, and lacks residues approximately 40-60 of alpha-glycophorin and its single asparagine-linked carbohydrate; it contains approximately 2 less O-glycosidically bound units whose structures are identical to those found in alpha-glycophorins. All these properties are characteristic of delta-glycophorin. The variant is related to alpha-glycophorin in the carboxyl-terminal region as shown by reaction with a specific antiserum. Sequence analyses of a mixture of chymotryptic peptides of a CNBr fragment of the variant glycophorin identified the sequence Val-His-Arg-Phe-Thr-Val-Pro-Glu-Ile-Thr-Leu-Ile-Ile that contains the junction point of delta- and alpha-glycophorins spanning residues 33-38/39 of delta-glycophorin and residues 71/72-77 of alpha-glycophorin. Sequence analysis of a mixture of CNBr fragments allowed us to conclude that the variant originates from delta-s- rather than delta-S-glycophorin. The quantity of the variant Dantu glycophorin when compared to alpha-glycophorin differed in the two individuals, the ratio being 2/1 in one individual's cells and 0.5/1 in the other's. This may reflect that the two donors belong to different varieties of Dantu phenotypes. Together, the evidence indicates that both donors' erythrocytes contain a (delta-alpha) variant glycophorin, whose amino terminus originates from delta-s-glycophorin and the carboxyl end from alpha-glycophorin with a junction point around residues 39 of delta- and 71 of alpha-glycophorins. The results suggest that the unique junction region may be characteristic of the Dantu phenotype.     

The role of carbohydrate in the blood group N-related epitopes recognised by three new monoclonal antibodies

The specificity of three new monoclonal anti-glycophorin antibodies, reacting preferentially with blood group N antigen, was characterized by means of untreated, enzymatically and chemically modified M and N glycoproteins. All antibodies recognized the NH₂-terminal Leu residue and its amino group, but differed in some other features, including the role of carbohydrate in the epitopes. One of the antibodies (631/3B4, IgM) showed an unusual two-directional dependence of activity on the degree of antigen desialylation. The progressive desialylation of N glycoprotein first caused a strongly increased binding to the epitope, followed by a complete loss of activity. The epitopes for the two remaining antibodies (648/4B5 and 650/4B5, both IgG₁) showed reactivity independent of sialylation, but dependent on the presence of Gal-GalNAc-units. Release of the disaccharide byO-glycanase treatment of N glycoprotein abolished its reactivity with both antibodies.     

Data bank of three-dimensional structures of disaccharides,a tool to build 3-D structures of oligosaccharides

This work presents the first part of a database of conformations for all the disaccharide fragments that are found inN-glycans. The conformational study of the five disaccharides found in the oligo-mannose type are presented here. For each disaccharide, several possible conformations are described. A method is presented to obtain realistic models of oligosaccharides using molecular mechanic methods. Analysis of some possible conformations of the oligo-mannose type glycan Man₆-GlcNAc₂ is given as an illustration of the possibilities.On post-doctoral leave from CERMAV-CNRS, Grenoble, France     

Exposure of major glycolipids in human Pk and p erythrocytes

The exposure of glycolipids in P^k and p red cells was studied by the galactose oxidase/ NaB²H₄ and galactose oxidase/NaB³H₄ surface labeling techniques. The major glycolipid in P^k cells, ceramide trihexoside was efficiently labeled when high amounts of galactose oxidase were used. In contrast, the major glycolipid in p cells, ceramide dihexoside was not oxidized by galactose oxidase. However, minor components with longer oligosaccharide chains were readily labeled in p cells by the galactose oxidase/NaB³H₄ method.Abbreviations CDH ceramide dihexoside, LacCer - CTH ceramide trihexoside, GbOse₃Cer     

Two monoclonal antibodies highly specific for the blood group N determinant

Two monoclonal IgM antibodies, 179K and 35/5F, obtained following immunization of mice with A₂,MN or O,MN human erythrocytes, agglutinate NN and MN red cells strongly, and MM erythrocytes weakly. As shown by hemagglutination inhibition and solid phase ELISA, both antibodies are highly specific for the blood group N determinant. They react with N glycoprotein, its amino-terminal glycopeptides and with Ss glycoprotein (glycophorin B), which carries the blood group N determinant. They fail to react with M glycoprotein, M glycoprotein-derived glycopeptides, or with internal glycopeptides derived from N glycoprotein. Reaction of the antibodies with N glycoprotein is abolished by desialylation, periodate oxidation/borohydride reduction, orN-acetylation of the glycoprotein. Thus, the antibodies are specific for an epitope which includes sialylated oligosaccharide chain(s) and is located in the region of the amino-terminal leucine residue of N glycoprotein. MMU^– erythrocytes, lacking both blood group N and Ss glycophorin are non-reactive. Agglutination of MMU⁺ erythrocytes by the anti-N antibodies occursvia interaction with glycophorin B and correlates with the Ss phenotype of red cells MM,S erythrocytes are usually more strongly, agglutinated than MM,ss cells. The agglutination of MM erythrocytes decreases markedly as the pH is increased from 6 to 8, while agglutination of NN red cells is much less affected by shifts in pH over this range. As a result, both monoclonal antibodies are highly anti-N specific typing reagents when the agglutination assay is carried out at pH 8.     

The structure of a fucose-containingO-glycosidic carbohydrate chain of human platelet glycocalicin

The hydrazinolysis procedure currently used for the release ofN-glycosidic carbohydrate chains was applied to glycocalicin. The resulting mixture of oligosaccharide-alditols was fractionated by high-voltage paper electrophoresis into a neutral (5%) and several acidic fractions. The neutral compounds were passed over Bio-Gel P-4. SomeN-glycosidic oligosaccharide-alditols, of theN-acetyllactosamine type as well as of the oligomannoside type, were found to be present. However, oligosaccharide-alditols derived fromO-glycosidic carbohydrate chains were also found, indicating a partial cleavage of GalNAc1-OSer/Thr linkages under the hydrazinolysis conditions applied. One of the neutralO-glycosidic components was characterized, by 500-MHz¹H-NMR spectroscopy in combination with sugar analysis, as the following pentasaccharidealditol: In addition the afuco analogue of this compound was obtained.     

Lysis of erythrocytes byTrichomonas vaginalis

The in vitro hemolytic activity of 4 isolates ofTrichomonas vaginalis was investigated. Repetitive hemolysis assays of any one isolate showed cyclical fluctuations in hemolytic activity, varying over 24 hr of continuous culture. Maximal hemolytic activity was detected using trichomonads in the lag phase of the growth cycle. Investigations showed that hemolysis was a contact-dependent phenomenon and microscopic investigation of samples showed a significant correlation between hemolysis and attachment of erythrocytes to the trichomonad surface. Quantitative data from cytoadherence assays using [⁵¹Cr]-labeled erythrocytes were consistent with these observations. It is suggested that hemolytic activity is dependent upon adherence of red blood cells to the surface ofT. vaginalis.     

The purine metabolism of human erythrocytes

This review summarizes currently available information about a crucial part of erythrocyte metabolism, that is, purine nucleotide conversions and their relationships with other conversion pathways. We describe the cellular resynthesis, interconversion, and degradation of purine compounds, and also the regulatory mechanisms in the conversion pathways. We also mention purine metabolism disorders and their clinical consequences. The literature is fragmentary because studies have concentrated only on selected aspects of purine metabolism; hence the need for a synthetic approach. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 5, pp. 581–591.     

The structure of a trisialyl di-antennaryN-type glycopeptide obtained from rat plasma hemopexin

Rat hemopexin is a plasma glycoprotein that contains 18.3% carbohydrate consisting of onlyN-glycosidically-linked oligosaccharide chains. Glycopeptides obtained from hemopexin by Pronase^® digestion could be separated on Concanavalin A-Sepharose into three fractions. The lectin-binding fraction has been characterized as a mixture of monosialyl and disialyl di-antennary compounds ending inN-acetylneuraminic acid residues (2-6)-linked to galactose in the respective branches [Bernard N, Lombart C, Strecker G, Montreuil J, Van Halbeek H, Vliegenthart JFG (1983) Biochimie 65:185–92].The structures of the glycans in the Concanavalin A non-binding fractions were determined by a combination of methylation analysis and 500-MHz¹H-NMR spectroscopy. Some of them appeared to be tri-antennary glycans. However, the major component of these fractions possesses the following structure: This type of structure has been encountered before in some bovine blood coagulation factors as well as in rat -acid glycoprotein, but the¹H-NMR parameters for it are first reported here. Furthermore, by methylation analysis, the occurrence of the NeuAc2-8NeuAc disaccharide element was demonstrated in a minor part of the carbohydrate moiety of rat hemopexin. This element has also been reported previously for rat brain glycopeptides.     

Three-dimensional structure of the catalytic core of acetylxylan esterase from Trichoderma reesei: insights into the deacetylation mechanism

Acetylxylan esterase from Trichoderma reesei removes acetyl side groups from xylan. The crystal structure of the catalytic core of the enzyme was solved at 1.9 A resolution. The core has an alpha/beta/alpha sandwich fold, similar to that of homologous acetylxylan esterase from Penicillium purpurogenum and cutinase from Fusarium solani. All three enzymes belong to family 5 of the carbohydrate esterases and the superfamily of the alpha/beta hydrolase fold. Evidently, the enzymes have diverged from a common ancestor and they share the same catalytic mechanism. The catalytic machinery of acetylxylan esterase from T. reesei was studied by comparison with cutinase, the catalytic site of which is well known. Acetylxylan esterase is a pure serine esterase having a catalytic triad (Ser90, His187, and Asp175) and an oxyanion hole (Thr13 N, and Thr13 O gamma). Although the catalytic triad of acetylxylan esterase has been reported previously, there has been no mention of the oxyanion hole. A model for the binding of substrates is presented on the basis of the docking of xylose. Acetylxylan esterase from T. reesei is able to deacetylate both mono- and double-acetylated residues, but it is not able to remove acetyl groups located close to large side groups such as 4-O-methylglucuronic acid. If the xylopyranoside residue is double-acetylated, both acetyl groups are removed by the catalytic triad: first one acetyl group is removed and then the residue is reorientated so that the nucleophilic oxygen of serine can attack the second acetyl group.     

Description and application of an immunological detection system for analyzing glycoproteins on blots

By introducing the steroid hapten digoxigenin specifically into sugars, a sensitive detection system for glycoproteins on blots has been developed. Sugars are oxidized to obtain aldehyde groups, which then react with digoxigenin-succinyl--amido caproic acid hydrazide. A high-affinity antibody, conjugated to alkaline phosphatase, is used for the detection of the incorporated digoxigenin.This system allows the detection of nanogram-amounts of glycoproteins on blots, and it's specificity allows a clear distinction of a glycoprotein from a non-glycoprotein. In combination with endo- and exoglycosidases it is very useful for determining the type of carbohydrate linkage in a glycoprotein, and by varying the oxidation conditions, specific labeling of sialic acids and terminal galactoses can be achieved.     

The fine structure and photosynthetic cost of structural leaf variegation

The leaves of some plants display an optical patchiness on their upper side, displaying light- and dark-green areas with high and low reflectance, respectively. In this investigation, we studied the fine structure of the corresponding sectors and we asked whether the lost reflected light entails a photosynthetic cost to these leaves. Four species, i.e. Arum italicum, Ranunculus ficaria, Cyclamen hederifolium and Cyclamen persicum were investigated. Scanning electron microscope examination revealed that epidermal cells of light-green sectors of all species are more bulgy than corresponding cells of neighboring dark-green leaf sectors. The comparative anatomical study revealed that (i) epidermis thickness of the light-green areas and the number of mesophyll cell layers does not differ from those of the adjacent dark-green leaf sectors and (ii) palisade cells of light-green sectors are slightly larger and more loosely arranged, allowing a much higher percentage of intercellular air spaces. The latter histological feature seems to provide the structural basis for the different optical properties between the two leaf sectors. Contrary to expectations, net photosynthetic rates (expressed on a leaf area basis) were similar in the light-green and the dark-green areas of the two cyclamen species. Yet, in C. persicum net photosynthesis was higher in the light-green areas, if expressed on a dry mass basis. The small size of the light-green spots in the rest of the test plants precluded CO₂ assimilation measurements, yet maximum linear photosynthetic electron transport rates displayed no differences between the two sectors in all plants. Thus, the assumption of a photosynthetic cost in the light-green areas was not confirmed. On the contrary, a higher construction cost was evident in the dark-green areas of three species, displaying a significantly higher specific leaf mass, without any photosynthetic benefit. The results on net photosynthesis were compatible with leaf optical properties and pigment levels. Thus, in spite of the considerably higher reflectance of the light-green areas and their lower (yet normal for a green leaf) chlorophyll levels, corresponding differences in absorptance were slight. In addition, dry mass-based pigment contents in dark-green areas were higher, while chlorophyll a/b (in two species) and carotenoid/chlorophyll ratios (in three species) were lower, pointing to a shade adaptation in these sectors. We conclude that in variegated leaves of this kind, dark-green areas are more costly to build and probably less photosynthetically active. We argue that the high pigment contents of dark-green areas establish steep light gradients in the corresponding mesophyll, rendering deeper chloroplast layers more shade adapted.     

The applicability of hns and fimA primers for detecting Salmonella in bioaerosols associated with animal and municipal wastes

The ability to rapidly screen environmental samples for specific pathogens such as Salmonella spp., is of particular importance in molecular epidemiology. Although gene amplification reactions allow the rapid detection of microorganisms, the use of appropriate oligonucleotide primers targeted against specific microbial genes is critical for accurate detection specificity and sensitivity. Primers such as fimA and hns have been previously shown to be specific for pure cultures of Salmonella. However, the analysis of environmental samples requires post-amplification hybridizations to detect amplicons, since the presence of inhibitory environmental components reduces amplification efficiency of the target organism. These sensitive post-amplification approaches also enable the detection of spurious amplification from non-target sequences. Bioaerosols associated with animal facilities and municipal wastes contain a diverse array of pathogens including Clostridium spp. In our studies, hybridization sensor data revealed spurious amplification of clostridial species with Salmonella hns primers. Specificity checks using type cultures of Clostridium spp. revealed non-specific amplification by hns primers. These results suggest that fimA primers may be better suited to screen Salmonella-specific sequences in environmental samples, especially those obtained from animal and municipal waste facilities.     

Structural homology between glycophorins C and D of human erythrocytes

Glycophorin C (GPC) and D (GPD) are minor glycoproteins which are believed to be important for the structural integrity of the red cell membrane. We have investigated the structural relationship between these glycoproteins by both immunological and structural investigations: 1. A rabbit anti-serum produced against GPD reacts strongly with GPC and the abnormal glycoproteins of Gerbich: -2, -3 and Gerbich: -2,3 red cells, and recognizes most probably the homologous C-terminal portions of GPC and GPD. The two molecules however differ at their N-terminus. 2. One-dimensional mapping of the peptides obtained after tryptic, chymotryptic, V8 protease or acid cleavage of 125I-labelled GPC and GPD, indicated that GPC and GPD are structurally related but some differences were found indicating that additional peptides were generated from GPC. 3. The partial primary structure of GPD was determined. The sequencing data are consistent with the assumption that GPD represents an abridged version of GPC that comprises residues approximately 21/29-128 and exhibits a N-terminal residue that is blocked by an as yet undefined group.