Glycodelin A, Not Glycodelin S, Is Apoptotically Active

Abstract

Glycodelin, previously known as PP14 (placental protein-14), is a kernel lipocalin secreted by the glandular epithelium of the endometrium upon progesterone stimulation and by the seminal vesicles. The isoform of the protein present in female reproductive tissue, glycodelin A (GdA), and the male counterpart, glycodelin S (GdS), have identical amino acid sequences, but strikingly different N-linked glycans. It is well documented in literature that GdA is an immunosuppressive protein, and we have shown that this activity is due to its ability to induce apoptosis in activated T cells. The precise role of GdS in seminal plasma is not known. In this study, we report that GdS is not apoptotically active. We observe that the apoptotic activity requires the presence of sialic acid residues on the complex glycans, as in the case of GdA; however, complex glycans of GdS are nonsialylated. We have expressed the wild-type protein in Pichia pastoris, which does not add sialic acid to the secreted proteins, and confirmed our observations that the protein is apoptotically inactive in the non-sialylated form. Our results indicate that differential glycosylation modulates the function of the different glycodelin isoforms.

Analysis of the Role of Oligosaccharides in the Apoptotic Activity of Glycodelin A

“…High fucosylation and no sialylation (13) on GdS perhaps keep this region inaccessible. This hypothesis is supported by our observations that full-length glycodelin expressed in P. pastoris is inactive where there is a high level of glycosylation and a lack of sialylation (16,26).…”

“…Another function of GdA well documented in the literature is immunosuppression (3) and induction of apoptosis in T cells (4). Recent studies from our laboratory demonstrated clearly that GdS lacks apoptotic activity as well (26). It was therefore logical to determine whether the glycans on GdA also dictate its immunosuppressive characteristics.…”

“…It was therefore logical to determine whether the glycans on GdA also dictate its immunosuppressive characteristics. Enzymatic deglycosylation of GdA using peptide N-glycosidase F under native conditions failed (26); therefore, we resorted to recombinant DNA technology to address this question.…”

“…In brief, the full-length glycodelin cDNA with the secretory signal, obtained from the uterine endometrium (26), was cloned between BamHI and PstI into the shuttle vector pFASTBAC1 within the mini-Tn7 element. This vector was transformed into Escherichia coli DH10BAC cells, which harbor the bacmid (baculoviral genome)-carrying kanamycin resistance marker and an attachment site for the bacterial transposon Tn7 (mini-attTn7).…”

“…Observations of several research groups suggest that GdA is immunosuppressive in function (3), and previous studies from our laboratory indicate that this effect is due to its capacity to induce apoptosis in T cells (4). Interestingly, glycodelin S (GdS), another isoform of glycodelin synthesized by the seminal vesicles and accumulating in the seminal plasma, is not apoptogenic (26) even though both isoforms share the same amino acid sequence. As the glycans on both isoforms are different and as glycosylation has been demonstrated to play an important role in the contraceptive activity of GdA (13), it appeared logical to investigate the relevance of glycosylation to the apoptogenic function.…”