Wisteria floribunda agglutinin-binding glycan expression is decreased in endometriomata

Abstract

Background: Glycosylation is one of the most common post-translational modifications of eukaryotic proteins and is known to undergo dynamic changes in a wide range of biological processes. To date, however, the glycan expression profiles in endometriosis are largely unknown. The objective of the study was to identify the panel of glycans that were aberrantly expressed in endometriosis, a hormone-dependent disease.

Methods: The glycan expression profiles in primary cultured human endometriotic cyst stromal cells (ECSCs) and normal endometrial stromal cells (NESCs) were determined by lectin microarray analysis. Distribution of Wisteria floribunda agglutinin (WFA)-binding glycans in ovarian endometriotic cysts and eutopic proliferative phase endometrium were assessed by lectin histochemistry. The expressions of N-acetylgalactosaminyl transferases that synthesize WFA-binding glycans were evaluated in ECSCs and NESCs.

Results: We found that the levels of WFA-binding glycans were decreased in ECSCs. Lectin histochemistry revealed that WFA-binding glycans were decreased only in the stromal components of the ovarian endometriotic cysts, but not in the epithelial components, compared to the eutopic proliferative phase endometrium. The expressions of N-acetylgalactosaminyl transferases that synthesize WFA-binding glycans were downregulated in ECSCs.

Conclusions: Utilizing lectin microarray analysis and lectin histochemistry, we found that WFA-binding glycans were decreased in endometriosis. The synthetic enzymes of WFA-binding glycans were significantly downregulated in ECSCs. It is suggested that reduced expression of N-glycans with WFA-binding properties on ECSCs is a novel characteristics of endometriosis.

Figure 1

Unsupervised hierarchical clustering of ECSCs and NESCs. ECSCs (n = 9) and NESCs (n = 11) were clustered according to the glycan expression profiles with 45 lectin probes that meet the filtering criteria. Unsupervised hierarchical clustering analysis of these 45 lectins showed that the patterns of glycan expression in ECSCs were similar to those in NESCs as a whole, suggesting that diseased tissues in endometriosis retain the characteristics of their origin, namely the eutopic endometrium, concerning glycan expression profiles.

Figure 2

Distribution of WFA-binding glycans in ovarian endometriotic cysts and normal proliferative phase endometrium. Both the glandular epithelial cells and the stromal component of ovarian endometriotic tissues as well as the normal proliferative phase endometrium were positive for WFA-binding glycans with strong to weak intensity depending on the samples (hematoxylin & eosin staining, original magnification ×40). Staining intensity for WFA-binding glycans assessed by histochemical staining score is summarized in Table  2.

Figure 3

mRNA expression of N-acetylgalactosaminyl transferases in ECSCs and NESCs. The mRNA expressions of B3GALNT1, B3GALNT2, B4GALNT1, B4GALNT2, B4GALNT3, and B4GALNT4 in ECSCs (n = 7) and NESCs (n = 5) were evaluated by quantitative RT-PCR. *p < 0.025 and **p < 0.0001vs. NESCs (Student’s t test).

Figure 4

Protein levels of N-acetylgalactosaminyl transferases in ECSCs and NESCs. (A) Results of Western blot analysis. (B) Relative protein levels of N-acetylgalactosaminyl transferases in ECSCs (n=3) and NESCs (n=3). The levels of B3GALNT1, B3GALNT2, B4GALNT2, and B4GALNT3 were significant higher in NESCs compared to ECSCs. Whereas, the protein expressions of B4GALNT1 and B4GALNT4 in ECSCs and NESCs were under the detection levels. *p < 0.005 vs. NESCs (Student’s t test).