Malaria invasion of human erythrocytes

The invasion of human red blood cells (RBC) by plosmodiol merozoites is a key event during malaria infection, and the inhibition o f invasion is regarded as a crucial goal of malaria vaccine development. For Plasmodium falciparum it has been suggested that the red cell sialoglycoproteins, glycophorins A, B and C, are receptors for invasion and that O-linked or N-linked carbohydrate structures may be involved as receptor sites(1-3). However, recent evidence suggests that the role o f these sialoglycoproteins and carbohydrates may have been overestimated. In this article, Peter Hermentin discusses the contradictory findings and presents a revised model for the invasion process.     

Wright (a + b-) human erythrocytes and Plasmodium falciparum malaria

We find Wr(a + b-) erythrocytes of donor M. Fr., which appear to carry a rare glycophorin A variant, to be fully susceptible to invasion by nine isolates of Plasmodium falciparum. Thus we fail to confirm the previous publication on the refractoriness of these erythrocytes. In addition the serum of donor M. Fr., which is known to contain anti-Wrb directed against an epitope located on glycophorin A in close proximity to the erythrocyte membrane, was not found to inhibit P. falciparum invasion of blood group O Rh- red blood cells. Despite this, different lines of evidence still indicate that glycophorin A is one of the receptors for erythrocyte invasion by P. falciparum. The Wrb epitope, however, does not appear to represent a distinct receptor site, which is in contrast to previous suggestions.     

Plasmodium falciparum: carbohydrates as receptor sites of invasion

Monosaccharides, disaccharides, and trisaccharides were tested as inhibitors of the in vitro growth of Plasmodium falciparum (strain FCB). While certain monosaccharides (N-acetyl-D-glucosamine, D-mannose, and 3-O-methyl-D-glucose) proved to exhibit a toxic or reversibly retarding effect on the intraerythrocytic development of the parasite, the corresponding alpha- or beta-methylglycosides did not. Several methylglycosides, synthetic di- and tri-saccharides, and artificial blood group antigens were further tested for inhibitory effects on invasion of host red blood cells in vitro. The synthetic disaccharides beta DGlcNAc(1----4) alpha DManOMe and beta DGlcNAc(1----4) DGlcNAc (chitobiose) were good inhibitors of invasion at 10 mM concentration, whereas beta DGal(1----4)beta DGlcNAcOMe was negligibly inhibitory. The inhibition rate of N-acetyl-D-glucosamine, beta-glycosidically linked to bovine serum albumin (BSA) by an alipathic spacer, -(CH2)8CO-, was not enhanced, compared to the corresponding hapten, beta DGlcNAcO(CH2)8COOCH3. The inhibition rates of blood group A- and B-trisaccharide haptens, which were inhibitors of invasion, were also not significantly enhanced when coupled to BSA by way of the corresponding amide spacer, -(CH2)2NHCO(CH2)7CO-. A remarkable enhancement of the inhibition rate was, however, observed when beta DGal(1----3) alpha DGalNAcO(CH2)2NHCO(CH2)7COOCH3 (T-hapten) was coupled to BSA. A clear-cut decrease in the inhibition rates of different beta-glycosides of N-acetyl-D-glucosamine, beta DGlcNAcOR, was observed, depending on the nature of the aglycon R(p-nitrophenyl greater than -(CH2)8COOCH3 greater than -(CH2)2NHCO(CH2)2COOCH3 greater than -CH3). Also, p-nitrophenyl-alpha-D-glucopyranoside was a much better inhibitor of invasion than the corresponding methyl glycoside, alpha DGlcOMe, which was not inhibitory. The properties of the aglycon spacer, used for the covalent attachment of the carbohydrate to the carrier protein, may thus be crucial for the outcome of the inhibition rate.     

Hinge-thiol coupling of monoclonal antibody to silanized iron oxide particles and evaluation of magnetic cell depletion

Iron oxide particles of average size 0.5-1.5 microns, covered by a silane coat carrying amino groups (Bio-Mag, Advanced Magnetics, Boston), were derivatized by reaction with N-[(gamma-maleimidobutyryl)oxy]-succinimide (GMBS), N-hydroxysuccinimidyl iodoacetate (NHIA), 2-iminothiolane (2-It), or N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The derivatized particles were suitable for the reaction with sulfhydryl groups and subsequently coated with monoclonal antibodies (MoAbs) of different classes and isotypes (IgM, IgG1, IgG2a, IgG2b, IgG3) as well as polyclonal rabbit anti-mouse IgG (RAM). The antibodies were reduced by dithiothreitol (DTT) and covalently conjugated to the BioMag derivatives via liberated sulfhydryls of the hinge region. The observed conjugation ratios, expressed as protein/iron (micrograms/mg), could be reproducibly varied for optimization. These ratios were dependent on the type and amount of antibody offered for coupling to the derivatized particles, decreasing as follows: polyclonal = IgM greater than IgG2b greater than IgG2a = IgG3 greater IgG1. The conjugation ratios were also dependent on the type and amount of the spacer used to derivatize the BioMag particles, decreasing as follows: GMBS greater than NHIA greater than 2-It greater than SPDP. The magnetically responsive magnetite-antibody conjugates ("magneto-beads"), carrying MoAb BMA 081 (anti-CD8; IgG2a), MoAb BB10 (anti-CD10/CALLA; IgG2b), MoAb VIL-A1 (anti-CD10; IgM), and polyclonal RAM, coupled similarly via 3.6 mumol of GMBS spacer per mg of Fe, were further investigated with respect to a depletion effect on specific cell subsets. The rates of cell depletion were found to be strongly dependent on the individual characteristics of the antibody used.(ABSTRACT TRUNCATED AT 250 WORDS)     

The hypothetical N-glycan charge: a number that characterizes protein glycosylation

The production of recombinant glycoprotein therapeutics requirescharacterization of glycosylation with respect to the lot-to-lotconsistency. Here we introduce the ‘hypothetical N-glycancharge Z’ as a parameter that allows to characterize theprotein glycosylation in a simple, however, efficient manner. The hypothetical N-glycan charge of a given glycoprotein isdeduced from the N-glycan mapping profile obtained via HPAE-PAD.In HPAEC, N-glycans are clearly separated according to theircharge, i.e., their number of sialic acid residues, providingdistinct regions for neutral struc tures as well as for themono- di-, tri, and tetrasialylated N-glycans (Hermentin etal., 1992a). Z is defined as the sum of the products of the respective areas(A) in the asialo, monosialo, disialo, trisialo, tetrasialo,and pentasialo region, each multiplied by the correspondingcharge: Thus, a glycoprotein with mostly C4-4^* structures will provideZ400 (e.g., rhu EPO (CHO), Z=361), a glycoprotein carrying largelyC3-3^* structures will amount to Z300 (e.g., bovine fetuin, Z=290),a glycoprotein with mostly C2-2^* structures will have Z200 (e.g.,human serum transferrin, Z=207, or human plasma AT III, Z=180),and a glycoprotein carrying only high-mannose type or trunkatedstructures will provide Z0 (e.g., bovine pancreas ribonucleaseB, Z=15, and hen ovomucoid, Z=15, respectively). The determination of Z was validated in multiple repetitiveexperiments and proved to be highly accurate and reliable. Zmay therefore be regarded as a new and characteristic parameterfor protein N-glycosylation. high-performance anion-exchange chromatography (HPAEC) pulsed amperometric detection (PAD) HPAE-PAD human plasma recombinant expression CHO BHK interleukin 4-receptor erythropoietin fetuin transferrin thyroglobulin antithrombin ribonuclease ovomucoid orosomucoid ₁-acid glycoprotein fibrinogen ₁ T-glycoprotein ₁-antitrypsin ₁-antichymotrypsin ß₂-glycoprotein I thyroxin-binding globulin ₁B-glycoprotein 8S₃-glycoprotein haptoglobin hydrazinolysis PNGase F consistency clearance in vivo half-life     

The patterns of the complex- and oligomannose-type glycans of uromodulin (Tamm-Horsfall glycoprotein) in the course of pregnancy

Uromodulin was isolated from urine of three pregnant women. Urine of each donor was collected at subsequent stages of their pregnancy and at one month after gestation. Each batch of uromodulin was enzymatically N-deglycosylated and the released N-glycans were isolated, quantified and profiled by high-pH anion-exchange chromatography. In the course of pregnancy no significant changes were detected in the negative charge distribution stemming from sialic acid and sulfate residues on the complex-type carbohydrate chains of uromodulin. Furthermore, no significant changes in the molar ratio between Man₆GlcNAc₂ and Man₇GlcNAc₂ were found in the course of pregnancy, only uromodulin from non-pregnant periods showed small differences.     

The mapping by high-pH anion-exchange chromatography with pulsed amperometric detection and capillary electrophoresis of the carbohydrate moieties of human plasma alpha 1-acid glycoprotein.

The reducing oligosaccharides released from alpha 1-acid glycoprotein (AGP) by conventional hydrazinolysis have been analyzed by two different mapping techniques, using high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) and capillary electrophoresis (CE) with uv detection at 190 nm. The CE measurements proved about 4000 times more sensitive than the measurements by HPAE-PAD. The N-glycan pool was fractionated by Mono Q anion-exchange chromatography, and individual fractions so obtained were desialylated using Vibrio cholerae neuraminidase. The resulting asialo-N-glycans were further analyzed by HPAE-PAD, revealing 2 major, 4 intermediate, and 4 small peaks and at least 3 spikes, which counted for at least 13 different asialo-N-glycans. The carbohydrate structures were tentatively assigned by comparison of the Mono Q-separated N-glycans with the known AGP carbohydrate structures and known structures contained in a mapping database that allows structural assignment of N-glycans by mere comparison of retention times. In addition to the hitherto known AGP carbohydrate structures, we have tentatively identified a number of sulfated N-glycans that are currently being analyzed in more detail. We have also compared the glycan pools recovered from AGP using hydrazinolysis and glycopeptidase F (PNGase F). Approximately 40 distinct peaks could be detected in the hydrazinolysis-derived N-glycan pool by either technique (HPAE-PAD and CE), while about 30 distinct peaks were detected in the N-glycan pool derived by PNGase F digestion of the tryptic AGP digest of the same batch of AGP. These differences were attributed to an increased desialylation (approximately 3 mol%) during hydrazinolysis, based on the detection by HPAE-PAD and CE of free sialic acid and monosialylated oligosaccharides in the glycan pool derived by conventional hydrazinolysis. The integrity of the N-glycans' chitobiose core was examined by 500-MHz 1H NMR spectoscopy. The hydrazinolysis procedure could be optimized such that the hydrazinolysis-derived N-glycan pool was chromatographically essentially identical to the PNGase F-derived N-glycan pool. Hydrazinolysis proved best, with practically no loss of N-acetlylneuraminic acid and the closest resemblance to the PNGase F-derived N-glycan pool, using an automated apparatus. Notably, it was recognized that, in our hands, PNGase F digestion in the presence of sodium dodecyl sulfate resulted in partial desialylation of the liberated N-glycans.     

A strategy for the mapping of N-glycans by high-pH anion-exchange chromatography with pulsed amperometric detection.

We have evaluated the high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) with respect to its suitability to establish a carbohydrate mapping database that would enable carbohydrate structural analysis by mere comparison of retention times. The suitability of HPAE-PAD for carbohydrate structural analysis was ascertained by validation experiments. The retention times of distinct N-glycans, prepared and measured on different days, were shown to be highly reproducible, with a coefficient of variation (CV) of less than 0.5%, requiring less than 100 pmol of N-glycan per injection for reliable measurements. Including appropriate internal chromatographic standards, such as (Neu5Ac)1, (Neu5Ac)2, (Neu5Ac)3, and Neu5Gc, the HPAE-PAD method fulfills the analytical requirements with respect to accuracy, precision, reproducibility, and sensitivity. The N-glycan mapping database was established, using two optimized linear gradients "S" and "A" for sialylated and asialo N-glycans, respectively. Approximately 100 different N-glycans of known structure, which have thus far been measured and characterized, have entered our Lotus 1-2-3 mapping database. The efficiency of the database for structural determinations was tested, using the N-linked carbohydrates isolated from rhuEPO, expressed in BHK cells. Nine different sialylated N-glycans of rhuEPO (BHK) could be assigned with a deviation of less than +/- 0.5%, using gradient S, and six of the eight asialo N-glycans of rhuEPO (BHK) detected with gradient A could be assigned with an accuracy of less than +/- 1%, three of them even with an accuracy of less than 0.1%, providing the reliability of the established HPAE-PAD mapping database.