Novel Glycosidic Linkage in Aedes aegypti Chorion Peroxidase

Abstract

Aedes aegypti chorion peroxidase (CPO) plays a crucial role in chorion hardening by catalyzing chorion protein cross-linking through dityrosine formation. The enzyme is extremely resistant to denaturing conditions, which seem intimately related to its posttranslational modifications, including disulfide bond formation and glycosylation. In this report, we have provided data that describe a new type of glycosylation in CPO, where a mannose is linked to the N-1 atom of the indole ring of Trp residue. Through liquid chromatography/electrospray ionization/tandem mass spectrometry and de novo sequencing of CPO tryptic peptides, we determined that three of the seven available Trp residues in mature CPO are partially (40 -50%) or completely mannosylated. This conclusion is based on the following properties of the electrospray ionization/tandem mass spectrometry spectra and the enzymatic reaction of these peptides Glycosylation is the most abundant post-translational modification in proteins. Based on the linkage of oligosaccharides with amino acid residues on proteins, O-glycosylation and N-glycosylation are the two best known types and have been extensively studied (1). In O-glycosylation, sugar is attached to the hydroxyl group of serine, threonine, tyrosine, or other amino acid residues through a hydroxyl group. In N-glycosylation, oligosaccharide is linked to the peptide through N-acetylglucosamine and asparagine with a recognition sequence of Asn-X-Ser/Thr, where X can be any amino acid except for proline. During the past 10 years, a new type of protein glycosylation involving attachment of an ␣-mannose to the C-2 carbon of the indole ring of Trp residues in proteins has been reported (2-4). It is termed C-glycosylation because this linkage is formed via a C-C bond. C-mannosylation has been proved to occur in a number of mammalian proteins, such as RNase2, interleukin-12, complements, properdin, thrombospondin, erythropoietin receptor, mucins, and a bovine lens protein (2-11, 13-16). A Trp-X-X-Trp (WXXW) motif seems to serve as the specificity determinant for C-mannosylation, in which the first Trp residue is mannosylated. In addition, it has been demonstrated that the C-mannosylation is an enzyme-catalyzed event (17). The potential function of the C-mannosylation has also been discussed in a recent report (15).In our study dealing with mosquito chorion hardening, we identified a specific chorion peroxidase (CPO) 2 that displayed extremely high physical stability under a number of denaturing conditions. For example, the enzyme remains active for months in 1-5% SDS (18).CPO undergoes extensive post-translational modifications, including proteolytic processing and glycosylation (19). Recently, we have determined the N-glycosylation site and N-glycan structures in CPO (20). In our current study dealing with the characterization of CPO post-translational modifications, we determined that some Trp residues in CPO were mannosylated. Further analysis of their MS/MS spectra determined that the mannosyl moiety is not lin...

A Glycosylated, Labionin-Containing Lanthipeptide with Marked Antinociceptive Activity

“…While N-glycosylindoles are encountered in microbial [e.g., staurosporins, 16 rebeccamycins, 17 peroxidase from the mosquito Aedes aegypti, the main vector of dengue and yellow fever, in which α-D-mannose is covalently connected via the N-1 atom of the indole ring. 20,21 NAI-112 Gene Locus. The Actinoplanes DSM24059 locus includes seven genes likely involved in NAI-112 biosynthesis.…”

A role of glycosyl moieties in the stabilization of bitter gourd (Momordica charantia) peroxidase

“…Some earlier workers have already shown that the presence of carbohydrate residues in proteins caused resistance to inactivation mediated urea [33]. In a recent study, Li et al [34] has reported that a peroxidase from Aedes aegypti chorion is extremely resistant to inactivation induced by various denaturing agents [34]. The carbohydrate part of the protein thus contributes towards stabilization of protein against denaturants [35].…”

Glycosylated Hydroxytryptophan in a Mussel Adhesive Protein from Perna viridis

“…Two ions with masses of 367 and 529 Da eluting between 22 and 24 min were detected; the latter decomposed readily to 367 through a loss of 162 Da (hexose) (supplemental Fig. S4), suggesting a cleavage that is typical of O-and N-linked hexoses (20,21). The 367-Da ion and its fragmentation, most notably the 120-Da loss to m/z 247, are identical with standard C 2 -[␣-D-mannopyranosyl]-tryptophan (22,23) (Fig.…”

“…It was first reported in pancreatic ribonuclease and since then in over 47 other proteins including notably thrombospondin and complement proteins with the sequence motif WX 1 X 2 W (20,(22)(23)(24)(25)(26). Although the function of this modification remains elusive, mannosylation is known to render the tryptophan more polar and solvent-accessible (23).…”