Maria Panico

Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito

Malaria is transmitted from vertebrate host to mosquito vector by mature sexual blood-living stages called gametocytes,. Within seconds of ingestion into the mosquito bloodmeal, gametocytes undergo gametogenesis. Induction requires the simultaneous exposure to at least two stimuli in vitro: a drop in bloodmeal temperature to 5 °C below that of the vertebrate host, and a rise in pH from 7.4 to 8.0–8.2 (refs 1, 4). In vivo the mosquito bloodmeal has a pH of between 7.5 and 7.6 (refs 5, 6). It is thought that in vivo the second inducer is an unknown mosquito-derived gametocyte-activating factor,,. Here we show that this factor is xanthurenic acid. We also show that low concentrations of xanthurenic acid can act together with pH to induce gametogenesis in vitro. Structurally related compounds are at least ninefold less effective at inducing gametogenesis in vitro. In Drosophila mutants with lesions in the kynurenine pathway of tryptophan metabolism (of which xanthurenic acid is a side product), no alternative active compound was detected in crude insect homogenates. These data could form the basis of the rational development of new methods of interrupting the transmission of malaria using drugs or new refractory mosquito genotypes to block parasite gametogenesis.

Structural Analysis of the Oligosaccharides Derived from Glycodelin, a Human Glycoprotein with Potent Immunosuppressive and Contraceptive Activities

Glycodelin, also known as placental protein 14 (PP14) or progesterone-associated endometrial protein (PAEP), is a human glycoprotein with potent immunosuppressive and contraceptive activities. In this paper we report the first characterization of glycodelin-derived oligosaccharides. Using strategies based upon fast atom bombardment and electrospray mass spectrometry we have established that glycodelin is glycosylated at Asn-28 and Asn-63. The Asn-28 site carries high mannose, hybrid and complex-type structures, whereas the second site is exclusively occupied by complex-type glycans. The major non-reducing epitopes in the complex-type glycans are: Galβ1-4GlcNAc (lacNAc), GalNAcβ1-4GlcNAc (lacdiNAc), NeuAcα2-6Galβ1-4GlcNAc (sialylated lacNAc), NeuAcα2-6GalNAcβ1-4GlcNAc (sialylated lacdiNAc), Galβ1-4(Fucα1-3)GlcNAc (Lewis), and GalNAcβ1-4(Fucα1-3)GlcNAc (lacdiNAc analogue of Lewis). It is possible that the oligosaccharides bearing sialylated lacNAc or lacdiNAc antennae may manifest immunosuppressive effects by specifically blocking adhesive and activation-related events mediated by CD22, the human B cell associated receptor. Oligosaccharides with fucosylated lacdiNAc antennae have previously been shown to potently block selectin-mediated adhesions and may perform the same function in glycodelin. The potent inhibitory effect of glycodelin on initial human sperm-zona pellucida binding is consistent with our previous suggestion that this cell adhesion event requires a selectin-like adhesion process. This result also raises the possibility that a convergence between immune and gamete recognition processes may have occurred in the types of carbohydrate ligands recognized in the human.

Meningococcal pilin: a glycoprotein substituted with digalactosyl 2,4-diacetamido-2,4,6-trideoxyhexose

Neisseria meningitidis pili are filamentous protein structures that are essential adhesins in capsulate bacteria. Pili of adhesion variants of meningococcal strain C311 contain glycosyl residues on pilin (PilE), their major structural subunit. Despite the presence of three potential N‐linked glycosylation sites, none appears to be occupied in these pilins. Instead, a novel O‐linked trisaccharide substituent, not previously found as a constituent of glycoproteins, is present within a peptide spanning amino acid residues 45 to 73 of the PilE molecule. This structure contains a terminal 1‐4‐linked digalactose moiety covalently linked to a 2,4‐diacetamido‐2,4,6‐trideoxyhexose sugar which is directly attached to pilin. Pilins derived from galactose epimerase (galE) mutants lack the digalactosyl moiety, but retain the diacetamidotrideoxyhexose substitution. Both parental (#3) pilins and those derived from a hyper‐adherent variant (#16) contained identical sugar substitutions in this region of pilin, and galE mutants of #3 were similar to the parental phenotype in their adherence to host cells. These studies have confirmed our previous observations that meningococcal pili are glycosylated and provided the first structural evidence for the presence of covalently linked carbohydrate on pili. In addition, they have revealed a completely novel protein/saccharide linkage.

Mass spectrometry of carbohydrate-containing biopolymers

Publisher Summary This chapter discusses practical aspects of fast atom bombardment–mass spectrometry (FAB–MS) and electrospray–mass spectrometry (ES–MS), including data interpretation, and describes examples of experimental strategies incorporating all three mass spectrometric methods, which are best suited to the analysis of glycoproteins, complex carbohydrates (including glycolipids), and glycosaminoglycans, respectively. In ES–MS technique a stream of liquid containing the sample of interest is injected directly into the ES source where the sample molecules are stripped of solvent, leaving them as multiply charged species whose charges reflect the number of functional groups that can be protonated (positive ion mode) or deprotonated (negative ion mode) at the pH of the carrier liquid. Two fragmentation pathways dominate carbohydrate FAB-MS, namely, A-type cleavage and β cleavage. A-type cleavage occurs on the nonreducing side of glycosidic bonds to give oxonium-type fragment ions and is the dominant pathway for permethyl and peracetyl derivatives. Cleavage is favored at amino sugar residues. A-type fragment ions are extremely helpful for structure assignments. β cleavage also involves glycosidic fission but, because a hydrogen transfer accompanies bond breakage, no charge is produced at the actual cleavage site as occurs during A-type cleavage.

Identification of Major Outer Surface Proteins of Streptococcus agalactiae

ABSTRACT To identify the major outer surface proteins of Streptococcus agalactiae (group B streptococcus), a proteomic analysis was undertaken. An extract of the outer surface proteins was separated by two-dimensional electrophoresis. The visualized spots were identified through a combination of peptide sequencing and reverse genetic methodologies. Of the 30 major spots identified as S. agalactiae specific, 27 have been identified. Six of these proteins, previously unidentified in S. agalactiae, were sequenced and cloned. These were ornithine carbamoyltransferase, phosphoglycerate kinase, nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase, purine nucleoside phosphorylase, enolase, and glucose-6-phosphate isomerase. Using a gram-positive expression system, we have overexpressed two of these proteins in an in vitro system. These recombinant, purified proteins were used to raise antisera. The identification of these proteins as residing on the outer surface was confirmed by the ability of the antisera to react against whole, live bacteria. Further, in a neonatal-animal model system, we demonstrate that some of these sera are protective against lethal doses of bacteria. These studies demonstrate the successful application of proteomics as a technique for identifying vaccine candidates.

Human sperm binding is mediated by the sialyl-Lewis(x) oligosaccharide on the zona pellucida.

Fertilization in humans is initiated by binding of spermatozoa to a selectin ligand on the egg’s extracellular matrix. Human fertilization begins when spermatozoa bind to the extracellular matrix coating of the oocyte, known as the zona pellucida (ZP). One spermatozoan then penetrates this matrix and fuses with the egg cell, generating a zygote. Although carbohydrate sequences on the ZP have been implicated in sperm binding, the nature of the ligand was unknown. Here, ultrasensitive mass spectrometric analyses revealed that the sialyl-Lewisx sequence [NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc], a well-known selectin ligand, is the most abundant terminal sequence on the N- and O-glycans of human ZP. Sperm-ZP binding was largely inhibited by glycoconjugates terminated with sialyl-Lewisx sequences or by antibodies directed against this sequence. Thus, the sialyl-Lewisx sequence represents the major carbohydrate ligand for human sperm-egg binding.

The myotropic and plasma-calcium modulating effects of calcitonin gene-related peptide (CGRP).

Human and rat calcitonin gene-related peptides cause a dose-related contraction of guinea pig ileum, which is antagonised by an anti-histamine, mepyramine, and an anticholinergic compound, hyoscine. Both peptides also cause a positive inotropic and a positive chronotropic effect in the rat isolated auricle and these responses are antagonised by propranolol, a B adrenoceptor blocker. Further, the peptides lower plasma calcium levels in both rats and rabbits in a dose-related manner resembling calcitonin; in the rabbit, but not in the rat, the initial calcium lowering effect is succeeded by hypercalcaemia at higher doses, while in the chick, only the parathyroid hormone-like calcium-raising effect is seen.

Molecular characterization of the surface layer proteins from Clostridium difficile

Many bacteria express a surface‐exposed proteinaceous layer, termed the S‐layer, which forms a regular two‐dimensional array visible by electron microscopy. Clostridium difficile is unusual in expressing two S‐layer proteins (SLPs), which are of varying size in a number of strains. In an approach combining molecular biology with mass spectrometric sequencing strategies, we have identified the structural gene (slpA) for the S‐layer from three strains of C. difficile. Both proteins are derived from a common precursor, and processing involves the removal of a signal peptide and a second cleavage to release the two mature SLPs. To our knowledge, this is the first example in which two SLPs have been shown to derive from a single gene product through post‐translational processing, rather than from the expression of separate genes. The higher molecular weight (MW) SLP is highly conserved among the three strains, whereas the lower MW SLP shows considerable sequence diversity, reflecting the results from Western blotting. The high‐MW SLP shows weak homology to N‐acetyl muramoyl‐l‐alanine amidase from Bacillus subtilis, and both the native SLP from C. difficile and a recombinant protein expressed in Escherichia coli were found to display amidase activity by zymography. The high‐MW SLPs showed evidence of glycosylation, whereas the lower MW proteins did not. A family of genes with sequence homology to the amidase domain of the high‐MW SLP was identified in the C. difficile strain 630 genome, some of which are located in the same region of the genome as slpA and were shown by reverse transcription–polymerase chain reaction (RT–PCR) analysis to be transcribed.

Gender-specific Glycosylation of Human Glycodelin Affects Its Contraceptive Activity

We have recently demonstrated that a human amniotic fluid-derived glycoprotein, glycodelin-A (GdA; previously known as PP14 or PAEP), potently inhibits gamete binding in an established sperm-egg binding system and expresses immunosuppressive activities directed against a variety of different immune cell types. GdA has high mannose-, hybrid-, and complex-type biantennary oligosaccharides including structures with fucosylated or sialylated N,N′-diacetyllactosediamine (GalNAcβ1-4GlcNAc) sequences, which are rare in other human glycoproteins. We now report the characterization of glycodelin-S (GdS). This is a human seminal plasma glycoprotein that is immunologically indistinguishable from GdA, but unlike the latter, does not inhibit human sperm-zona pellucida binding under hemizona assay conditions. Analysis of the N-glycans of GdS by mass spectrometry revealed that all glycoforms of GdS are different from those of GdA. GdS glycans are unusually fucose-rich, and the major complex-type structures are biantennary glycans with Lewisx (Galβ1-4(Fucα1-3)GlcNAc) and Lewisy (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc) antennae. It is probable that these highly fucosylated epitopes contribute to the immunosuppressive activity of human seminal plasma and to the low immunogenicity of sperm. This study provides the first evidence for gender-specific glycosylation that may serve to regulate key processes involved in human reproduction.

Glycomic profiling of cells and tissues by mass spectrometry: fingerprinting and sequencing methodologies.

Over the past decade, rapid, high-sensitivity mass spectrometric strat-egies have been developed and optimized for screening for the types of N- and O-glycans present in a diverse range of biological material, including secretions, cell lines, tissues, and organs. These glycomic strategies are based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass fingerprinting of permethylated derivatives, combined with electrospray (ES) or MALDI tandem mass spectrometry (MS/MS) sequencing and gas chromatography (GC)-MS linkage analysis, complemented by chemical and enzymatic degradations. Protocols for these methods are described in the first part of this chapter. Glycomic experiments yield large volumes of MS data, and interpretation of the resulting spectra remains a time-consuming bottleneck in the process. In the second part of this chapter, we describe the use and operation of a mass spectral viewer program capable of displaying and automatically labeling spectra arising from MALDI fingerprinting of N-glycans.