Impact of bacterial vaginosis, as assessed by nugent criteria and hormonal status on glycosidases and lectin binding in cervicovaginal lavage samples

The objective of this study was to evaluate the impact of hormonal status and bacterial vaginosis (BV) on the glycosidases present and glycosylation changes as assessed by lectin binding to cervicovaginal lavage constituents. Frozen cervicovaginal lavage samples from a completed study examining the impact of reproductive hormones on the physicochemical properties of vaginal fluid were utilized for the present study. In the parent study, 165 women were characterized as having BV, intermediate or normal microflora using the Nugent criteria. The presence of glycosidases in the samples was determined using quantitative 4-methyl-umbelliferone based assays, and glycosylation was assessed using enzyme linked lectin assays (ELLA). Women with BV had elevated sialidase, α-galactosidase, β-galactosidase and α-glucosidase activities compared to intermediate or normal women (P<0.001, 0.003, 0.006 and 0.042 respectively). The amount of sialic acid (Sambucus nigra, P = 0.003) and high mannose (griffithsin, P<0.001) were reduced, as evaluated by lectin binding, in women with BV. When the data were stratified according to hormonal status, α-glucosidase and griffithsin binding were decreased among postmenopausal women (P<0.02) when compared to premenopausal groups. These data suggest that both hormonal status and BV impact the glycosidases and lectin binding sites present in vaginal fluid. The sialidases present at increased levels in women with BV likely reduce the number of sialic acid binding sites. Other enzymes likely reduce griffithsin binding. The alterations in the glycosidase content, high mannose and sialic acid binding sites in the cervicovaginal fluid associated with bacterial vaginosis may impact susceptibility to viruses, such as HIV, that utilize glycans as a portal of entry

Studying the effects of reproductive hormones and bacterial vaginosis on the glycome of lavage samples from the cervicovaginal cavity

The cervicovaginal fluid (CVF) coating the vaginal epithelium is an important immunological mediator, providing a barrier to infection. Glycosylation of CVF proteins, such as mucins, IgG and S-IgA, plays a critical role in their immunological functions. Although multiple factors, such as hormones and microflora, may influence glycosylation of the CVF, few studies have examined their impact on this important immunological fluid. Herein we analyzed the glycosylation of cervicovaginal lavage (CVL) samples collected from 165 women under different hormonal conditions including: (1) no contraceptive, post-menopausal, (2) no contraceptive, days 1-14 of the menstrual cycle, (3) no contraceptive, days 15-28 of the menstrual cycle, (4) combined-oral contraceptive pills for at least 6 months, (5) depomedroxyprogesterone acetate (Depo-Provera) injections for at least 6 months, (6) levonorgestrel IUD for at least 1 month. Glycomic profiling was obtained using our lectin microarray system, a rapid method to analyze carbohydrate composition. Although some small effects were observed due to hormone levels, the major influence on the glycome was the presence of an altered bacterial cohort due to bacterial vaginosis (BV). Compared to normal women, samples from women with BV contained lower levels of sialic acid and high-mannose glycans in their CVL. The change in high mannose levels was unexpected and may be related to the increased risk of HIV-infection observed in women with BV, as high mannose receptors are a viral entry pathway. Changes in the glycome were also observed with hormonal contraceptive use, in a contraceptive-dependent manner. Overall, microflora had a greater impact on the glycome than hormonal levels, and both of these effects should be more closely examined in future studies given the importance of glycans in the innate immune system

Discovery of Inhibitors of Leishmania β-1,2-Mannosyltransferases Using a Click-Chemistry-Derived Guanosine Monophosphate Library

Leishmania spp. are a medically important group of protozoan parasites that synthesize a novel intracellular carbohydrate reserve polymer termed mannogen. Mannogen is a soluble homopolymer of β-1,2-linked mannose residues that accumulates in the major pathogenic stages in the sandfly vector and mammalian host. While several steps in mannogen biosynthesis have been defined, none of the enzymes have been isolated or characterized. We report the development of a simple assay for the GDP-mannose–dependent β-1,2-mannosyltransferases involved in mannogen synthesis. This assay utilizes octyl α-d-mannopyranoside to prime the formation of short mannogen oligomers up to 5 mannose residues. This assay was used to screen a focussed library of 44 GMP-triazole adducts for inhibitors. Several compounds provided effective inhibition of mannogen β-1,2-mannosyltransferases in a cell-free membrane preparation. This assay and inhibitor compounds will be useful for dissecting the role of different mannosyltransferases in regulating de novo biosynthesis and elongation reactions in mannogen metabolism

Bioinformatics and molecular modeling in glycobiology

The field of glycobiology is concerned with the study of the structure, properties, and biological functions of the family of biomolecules called carbohydrates. Bioinformatics for glycobiology is a particularly challenging field, because carbohydrates exhibit a high structural diversity and their chains are often branched. Significant improvements in experimental analytical methods over recent years have led to a tremendous increase in the amount of carbohydrate structure data generated. Consequently, the availability of databases and tools to store, retrieve and analyze these data in an efficient way is of fundamental importance to progress in glycobiology. In this review, the various graphical representations and sequence formats of carbohydrates are introduced, and an overview of newly developed databases, the latest developments in sequence alignment and data mining, and tools to support experimental glycan analysis are presented. Finally, the field of structural glycoinformatics and molecular modeling of carbohydrates, glycoproteins, and protein–carbohydrate interaction are reviewed

Molecular imaging using fluorescent lectins permits rapid endoscopic identification of dysplasia in Barrett's esophagus.

Barrett's esophagus is an example of a pre-invasive state, for which current endoscopic surveillance methods to detect dysplasia are time consuming and inadequate. The prognosis of cancer arising in Barrett's esophagus is improved by early detection at the stage of mucosal carcinoma or high-grade dysplasia. Molecular imaging methods could revolutionize the detection of dysplasia, provided they permit a wide field of view and highlight abnormalities in real time. We show here that cell-surface glycans are altered in the progression from Barrett's esophagus to adenocarcinoma and lead to specific changes in lectin binding patterns. We chose wheat germ agglutinin as a candidate lectin with clinical potential. The binding of wheat germ agglutinin to human tissue was determined to be specific, and we validated this specific binding by successful endoscopic visualization of high-grade dysplastic lesions, which were not detectable by conventional endoscopy, with a high signal-to-background ratio of over 5

Molecular imaging using fluorescent lectins permits rapid endoscopic identification of dysplasia in Barrett's esophagus.

Barrett's esophagus is an example of a pre-invasive state, for which current endoscopic surveillance methods to detect dysplasia are time consuming and inadequate. The prognosis of cancer arising in Barrett's esophagus is improved by early detection at the stage of mucosal carcinoma or high-grade dysplasia. Molecular imaging methods could revolutionize the detection of dysplasia, provided they permit a wide field of view and highlight abnormalities in real time. We show here that cell-surface glycans are altered in the progression from Barrett's esophagus to adenocarcinoma and lead to specific changes in lectin binding patterns. We chose wheat germ agglutinin as a candidate lectin with clinical potential. The binding of wheat germ agglutinin to human tissue was determined to be specific, and we validated this specific binding by successful endoscopic visualization of high-grade dysplastic lesions, which were not detectable by conventional endoscopy, with a high signal-to-background ratio of over 5

Polysialic acid bioengineering of cancer and neuronal cells by N-acyl sialic acid precursor treatment

Peer reviewed: YesNRC publication: Ye