Ewa M. Kratz

Alterations of branching and differential expression of sialic acid on alpha-1-acid glycoprotein in human seminal plasma

BACKGROUND The degree of branching and types of fucosylation of glycans on alpha(1)-acid glycoprotein (AGP) have been found to be associated with alpha(1)-acid glycoprotein concentrations in human seminal plasma. The glycosylation pattern of alpha(1)-acid glycoprotein in seminal plasma obtained from men living in infertile couples can undergo alterations in relation to sperm analysis and/or alpha(1)-acid glycoprotein concentrations. METHODS The glycosylation of alpha(1)-acid glycoprotein was studied upon the reactivity with specific lectins by crossed affinity immunoelectrophoresis (concanavalin A), and by glycoprotein lectin immunosorbent assay (Maackia amurensis and Sambucus nigra lectins), as well as high pH anion-exchange chromatography with pulsed amperometric detection. RESULTS Nonsignificant differences in alpha(1)-acid glycoprotein glycan branching and degree of its sialylation were observed among the AGP derived from seminal plasmas in relation to spermiogram and sperm morphology. However, significant concentration-dependent differences were found in extent of branching and type of sialylation. CONCLUSIONS The presence in seminal plasma of high concentrations of aberrantly glycosylated AGP molecules might be indicative for a chronic inflammatory condition in the reproductive tract, and can be used as additional tool to subdivide the seminal plasmas of men living in infertile couples.

Seminal plasma glycoproteins in male infertility and prostate diseases: is there a chance for glyco-biomarkers?

Context: Disturbed protein–carbohydrate interactions may underlie the molecular mechanism of some diseases of the male reproductive tract, including infertility and prostate diseases. Objective: To summarize the current knowledge on the glycosylation patterns of glycodelin-S, fibronectin, prostate-specific antigen, and α1-acid glycoprotein. Results: Some rare glycoepitopes have been found in seminal plasma glycoproteins: high-mannose and polylactosamine-type glycans, and N-glycans containing N-acetyl-galactosamine. The glycosylation profiles occur altered in pathological conditions. Conclusion: Further detailed studies may lead up to indicate the biomarkers useful in the management of male reproductive tract disorders.

Dental caries related to plasma IgG and alpha1-acid glycoprotein

This study was aimed at determining whether dental caries is associated with induction of the systemic immune system or cytokine response. For this purpose, 85 children from Den Pasar, Bali, Indonesia, aged 6–7 years, were examined clinically and blood plasma was obtained via finger puncture. The concentrations of the acute-phase protein α1-acid glycoprotein (AGP), total IgG and the specific IgG and IgM immunoglobulins against Streptococcus mutans were determined. Immunoelectrophoresis was used for the determination of the AGP concentration and ELISA for IgG and IgM detection. The mean dmft of the whole group was 8.8 ± 2.9, the mean number of infected pulps was 3.9 ± 2.2 and the mean number of abscesses was 0.5 ± 0.8. The plasma concentration of AGP ranged between 0.13 and 1.6 mg/ml serum (mean 0.86 ± 0.26 mg/ml). Stepwise regression analysis revealed that the concentration of IgG against S. mutans (log-transformed) was significantly correlated with dmft (adjusted r2 = 0.083, standardized β coefficient = 0.31, p = 0.008). When the concentration AGP was included in the model the correlation improved significantly (for IgG: adjusted r2 = 0.157, standardised β coefficient = 0.36, p = 0.002; for AGP: β coefficient = –0.30, p = 0.009). The results suggest a relationship between caries and systemic parameters of inflammation. On the basis of this, severe caries might have consequences on the general health of the subject.

Changes in glycosylation of human blood plasma chitotriosidase in patients with type 2 diabetes

Human blood plasma chitotriosidase (CHIT1) is a glycoprotein with chitinolytic activity with not fully elucidated biological function. Its increased level is observed in type 2 diabetes mellitus (T2DM) and is associated with development of diabetic complications. The CHIT1 glycosylation profile and degree is still poorly studied and never investigated in T2DM. Therefore the aim of the present study was to examine the association between glycosylation profile and degree and diabetes with accompanying nephropathy. In blood plasma of 28 patients with T2DM and 11 healthy subjects the CHIT1 concentration and specific activity were examined. The profile and degree of CHIT1 glycosylation were determined by lectin-ELISA using lectins specific to O-glycans (Jacalin, MPL, VVL) and sialo-specific SNA and MAA. We revealed that both concentration and specific activity of CHIT1 significantly increased in T2DM, especially in nephropathy with elevated albuminuria. The relative reactivities with lectins, except Jacalin, decreased progressively with T2DM occurrence and albuminuria progression. The most significant differences were observed between control vs. albuminuric group (Micro and Macro). It is also possible that the observed differences in immunoblotting pattern in molecular masses of CHIT1 bands between T2DM patients and healthy subjects may be caused by the differences in degree of CHIT1 glycosylation. The analysis of CHIT1 glycosylation status and the determination of CHIT1 concentration together with its enzymatic activity in blood plasma might constitute additional valuable diagnosis tools for the evaluation the T2DM patients with accompanying nephropathy. Extension of the lectin panel specific to O-glycans occurs useful for the further research using microarray formats, which are expected to accelerate “lectin-based glycan profiling” of glycoproteins.

Terminal monosaccharide screening of synovial immunoglobulins G and A for the early detection of rheumatoid arthritis

The expressions of some terminal glycotopes of synovial immunoglobulins G, A, and M were analysed in relation to rheumatoid arthritis (RA) progression defined according to early and advanced radiological changes in patients’ hands. The relative amounts of terminal monosaccharides were determined by lectin-immunoblotting of immunoglobulin preparations using appropriate lectins able to recognize α2,6-linked (Sambucus nigra agglutinin) and α2,3-linked (Maackia amurensis agglutinin) sialic acid, galactose (Ricinus communis agglutinin I), N-acetylglucosamine (Griffonia simplicifolia agglutinin II) as well as α1,6-linked (Aleuria aurantia lectin), α1,3-linked (Lotus tetragonolobus agglutinin), and α1,2-linked (Ulex europaeus agglutinin) fucose. The results indicate differences between early and advanced RA stages in the terminal sugar exposition of synovial IgG and IgA, but not IgM. The galactose-deficient glycotope with exposed N-acetylglucosamine of the synovial 33.1-kDa IgG fragment appeared exclusively in the early stage of RA. In contrast, this glycotope of intact synovial IgG and IgA was present in both groups, although with higher proportions in advanced RA. The proportions of the sialyl and fucosyl determinants of intact synovial A and G immunoglobulins were clearly lower in the early RA group than in the advanced. The analysis of terminal oligosaccharide exposition in IgG, IgG fragments, and IgA present in the synovial fluid of RA patients might be applicable as a stage-specific marker in the diagnosis and therapy of RA patients.

Decreased melatonin levels and increased levels of advanced oxidation protein products in the seminal plasma are related to male infertility

Melatonin, an indolamine secreted by the pineal gland, is known as a powerful free-radical scavenger and wide-spectrum antioxidant. Therefore, the aim of this study was to correlate markers of oxidative protein damage (advanced oxidation protein products, AOPPs) and the total antioxidant capacity (TAC) with melatonin levels in the seminal plasma of men with azoospermia (n=37), theratozoospermia (n=29) and fertile controls (normozoospermia, n=37). Melatonin concentration was measured by radioimmunoassay. The levels of AOPP as well as TAC efficiency (determined by the ferric reducing antioxidant power, FRAP) were estimated by spectrophotometric methods. The concentration of melatonin and AOPP significantly differed in azoospermic (P<0.0001) and theratozoospermic (P<0.0001) patients versus fertile men, and correlated negatively (r=-0.33, P=0.0016). The TAC levels were significantly higher in azoospermia than in theratozoospermia (P=0.0022) and the control group (P=0.00016). In azoospermia, the AOPP concentration was also significantly higher than that observed in theratozoospermia (P=0.00029). Decreased levels of melatonin together with elevated AOPP altered the oxidative-antioxidative balance in the ejaculate, thereby reducing fertility. Therefore, melatonin and AOPP levels may serve as additional diagnostic markers of semen quality and male reproductive potential.

Comparison of haptoglobin and alpha₁-acid glycoprotein glycosylation in the sera of small cell and non-small cell lung cancer patients.

INTRODUCTION Cancer-related carbohydrate epitopes, which are regarded as potential diagnostic and prognostic biomarkers, are carried on the main acute phase proteins. It is not clear, however, if the glycosylation profile is similar in different glycoproteins, or it is protein specific to some extent. The aim of the study was to compare fucosylation, α2,3 sialylation and expression of sialyl-Lewisx epitopes (sLe(x)) in the serum as a whole, AGP and haptoglobin of small cell (SCLC) and non-small cell lung cancer (NSCLC) patients with respect to healthy subjects as well as the cancer stage and its histological type. MATERIAL AND METHODS Thirty-three NSCLC, 13 SCLC patients and 20 healthy volunteers were included in the study. Carbohydrate epitopes were detected by means of their reactivity with specific lectins and monoclonal anti-sLe(x) antibodies in direct or dual-ligand ELISA tests. RESULTS Significantly increased fucosylation was found in total serum in both cancer groups and in NSCLC haptoglobin. No difference was observed in SCLC haptoglobin or α₁-acid glycoprotein in both cancer groups. Also α2,3 sialylation was elevated in total serum, but not in α₁-acid glycoprotein. This type of sialylation was undetectable in haptoglobin by means of MAA reactivity, in both healthy and cancer subjects. Complete sLe(x) antigens were overexpressed in total NSCLC serum and SCLC AGP, and their level was considerably lowered in cancer haptoglobin. DISCUSSION Typical acute phase proteins, haptoglobin and AGP, exhibit different glycosylation profiles in lung cancer. Alterations observed in haptoglobin reflected the disease process better than those in AGP. Comparison of haptoglobin and AGP glycosylation to that observed in total serum suggests that some efficient carriers of disease-altered glycoproteins still remain unidentified.

Gelatinases and their tissue inhibitors are associated with oxidative stress: a potential set of markers connected with male infertility.

The expression and activity of matrix metalloproteinases (MMPs) may be regulated by oxidative stress in various pathophysiological processes; therefore, the aim of the present study was to analyse the associations between the expression of the gelatinases MMP-9 and MMP-2 and their tissue inhibitors TIMP-1, TIMP-2 and levels of total antioxidant capacity (TAC) and advanced oxidation protein products (AOPP) in seminal plasma prepared for artificial insemination. Levels of MMPs and TIMPs were evaluated using ELISA, whereas TAC and AOPP in the seminal plasma of 131 childless men and 38 fertile volunteers were determined spectrophotometrically. Seminal MMP-9 expression was higher in childless men than in fertile subjects, whereas there was no significant differences in MMP-2 expression between the analysed seminal groups. TIMP-1 and TIMP-2 expression was similar in all groups. However, TAC expression was significantly higher in infertile normozoospermic and oligozoospermic men and AOPP expression was higher in astheno-, oligo- and normozoospermic infertile patients than in fertile men. High AOPP, together with an increased MMP-9:TIMP-1 ratio alters the oxidative-antioxidative balance of the ejaculate, thereby reducing male fertility, and therefore these parameters may serve as additional diagnostic markers of semen quality and male reproductive potential.

Terminal Mannose Residues in Seminal Plasma Glycoproteins of Infertile Men Compared to Fertile Donors

The impact of seminal plasma components on the fertilization outcomes in humans is still under question. The increasing number of couples facing problems with conception raises the need for predictive biomarkers. Detailed understanding of the molecular mechanisms accompanying fertilization remains another challenge. Carbohydrate–protein recognition may be of key importance in this complex field. In this study, we analyzed the unique glycosylation pattern of seminal plasma proteins, the display of high-mannose and hybrid-type oligosaccharides, by means of their reactivity with mannose-specific Galanthus nivalis lectin. Normozoospermic infertile subjects presented decreased amounts of lectin-reactive glycoepitopes compared to fertile donors and infertile patients with abnormal semen parameters. Glycoproteins containing unveiled mannose were isolated in affinity chromatography, and 17 glycoproteins were identified in liquid chromatography-tandem mass spectrometry with electrospray ionization. The N-glycome of the isolated glycoproteins was examined in matrix-assisted laser desorption ionization mass spectrometry. Eleven out of 27 identified oligosaccharides expressed terminal mannose residues, responsible for lectin binding. We suggest that lowered content of high-mannose and hybrid type glycans in normozoospermic infertile patients may be associated with impaired sperm protection from preterm capacitation and should be considered in the search for new infertility markers.

Preliminary MALDI-TOF-MS analysis of seminal plasma N-glycome of infertile men

Glycosylation pattern within reproductive tract is now suggested to be involved in providing female immune tolerance for allograft sperm and developing embryo, but the information whether impaired glycosylation may influence male fertility potential is still limited. We have analyzed seminal plasma N-glycome in pooled samples derived from fertile and infertile men by means of MALDI-TOF/TOF tandem mass spectrometry. Among infertile subjects, normozoospermic, oligozoospermic, asthenozoospermic and oligoasthenozoospermic samples were obtained. Eighty-six oligosaccharides were identified in all the analyzed samples. Differences in the content of unique glycans: high mannose and hybrid type, lacking terminal sialic acid and highly fucosylated were found when samples derived from infertile subjects with different semen patterns were compared to the fertile control. The content of highly branched glycans was 3-fold elevated in normozoospermic infertile men, while the expression of highly fucosylated oligosaccharides was increased in asthenozoospermic, oligozoospermic and oligoasthenozoospermic samples. Sialylation of oligosaccharides was decreased in oligozoospermic, oligoasthenozoospermic and especially asthenozoospermic samples, but increased in infertile normozoospermic subjects. Altered glycosylation observed in seminal plasma may reflect similar changes in sperm surface glycoproteins, and may disturb sperm interaction with female immune system. We suggest that at least some cases of unexplained male infertility may be associated with impaired glycosylation.