Hannu Koistinen

Structural Analysis of the Oligosaccharides Derived from Glycodelin, a Human Glycoprotein with Potent Immunosuppressive and Contraceptive Activities

Glycodelin, also known as placental protein 14 (PP14) or progesterone-associated endometrial protein (PAEP), is a human glycoprotein with potent immunosuppressive and contraceptive activities. In this paper we report the first characterization of glycodelin-derived oligosaccharides. Using strategies based upon fast atom bombardment and electrospray mass spectrometry we have established that glycodelin is glycosylated at Asn-28 and Asn-63. The Asn-28 site carries high mannose, hybrid and complex-type structures, whereas the second site is exclusively occupied by complex-type glycans. The major non-reducing epitopes in the complex-type glycans are: Galβ1-4GlcNAc (lacNAc), GalNAcβ1-4GlcNAc (lacdiNAc), NeuAcα2-6Galβ1-4GlcNAc (sialylated lacNAc), NeuAcα2-6GalNAcβ1-4GlcNAc (sialylated lacdiNAc), Galβ1-4(Fucα1-3)GlcNAc (Lewis), and GalNAcβ1-4(Fucα1-3)GlcNAc (lacdiNAc analogue of Lewis). It is possible that the oligosaccharides bearing sialylated lacNAc or lacdiNAc antennae may manifest immunosuppressive effects by specifically blocking adhesive and activation-related events mediated by CD22, the human B cell associated receptor. Oligosaccharides with fucosylated lacdiNAc antennae have previously been shown to potently block selectin-mediated adhesions and may perform the same function in glycodelin. The potent inhibitory effect of glycodelin on initial human sperm-zona pellucida binding is consistent with our previous suggestion that this cell adhesion event requires a selectin-like adhesion process. This result also raises the possibility that a convergence between immune and gamete recognition processes may have occurred in the types of carbohydrate ligands recognized in the human.

Gender-specific Glycosylation of Human Glycodelin Affects Its Contraceptive Activity

We have recently demonstrated that a human amniotic fluid-derived glycoprotein, glycodelin-A (GdA; previously known as PP14 or PAEP), potently inhibits gamete binding in an established sperm-egg binding system and expresses immunosuppressive activities directed against a variety of different immune cell types. GdA has high mannose-, hybrid-, and complex-type biantennary oligosaccharides including structures with fucosylated or sialylated N,N′-diacetyllactosediamine (GalNAcβ1-4GlcNAc) sequences, which are rare in other human glycoproteins. We now report the characterization of glycodelin-S (GdS). This is a human seminal plasma glycoprotein that is immunologically indistinguishable from GdA, but unlike the latter, does not inhibit human sperm-zona pellucida binding under hemizona assay conditions. Analysis of the N-glycans of GdS by mass spectrometry revealed that all glycoforms of GdS are different from those of GdA. GdS glycans are unusually fucose-rich, and the major complex-type structures are biantennary glycans with Lewisx (Galβ1-4(Fucα1-3)GlcNAc) and Lewisy (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc) antennae. It is probable that these highly fucosylated epitopes contribute to the immunosuppressive activity of human seminal plasma and to the low immunogenicity of sperm. This study provides the first evidence for gender-specific glycosylation that may serve to regulate key processes involved in human reproduction.

A role for glycoconjugates in human development: the human feto-embryonic defence system hypothesis

The mechanisms underlying the protection of the human embryo/fetus from the maternal immune response are poorly understood. Substantial evidence indicates that carbohydrate recognition plays a primary role in the sequestration of leukocytes during inflammatory processes, lymphocyte homing, and initial gamete binding. Our previous studies suggest a possible convergence in the types of carbohydrate sequences recognized during initial human gamete binding and immune/inflammatory cell interactions. Our more recent findings indicate that oligosaccharides participating in such processes are also associated with soluble glycoconjugates found in the human placenta, amniotic fluid, and decidua. We theorize that such glycoconjugates may abrogate the maternal immune/inflammatory response by blocking the primary adhesive interactions required for the expression of such activities. Foreign embryonic cells may also be protected by surface expression of oligosaccharide sequences that suppress immune effector cell action in a manner not dependent upon classical major histocompatibility (MHC) recognition. Glycoconjugates expressing selectin ligands may also manifest a potent contraceptive effect that may also be beneficial for both the mother and the developing embryo/fetus. This hypothesis provides a preliminary framework for understanding how temporally and spatially restricted immunosuppressive effects could be expressed in utero that protect the human embryo/fetus during this period of human development.

Glycodelin-A interacts with fucosyltransferase on human sperm plasma membrane to inhibit spermatozoa-zona pellucida binding

Fertilization depends on successful binding of the spermatozoa to the zona pellucida of the oocyte. Glycodelin-A inhibits spermatozoa-zona pellucida binding. Previous data showed that glycodelin-A receptor(s) and zona pellucida protein receptor(s) on human spermatozoa are closely related. Using a chemical cross-linking approach, the glycodelin-A-sperm receptor complex was isolated. The receptor was identified to be fucosyltransferase-5 (FUT5) by mass spectrometry and confirmed with the use of anti-FUT5 antibodies. Sperm FUT5 was an externally oriented integral membrane protein in the acrosomal region of human spermatozoa. Biologically active FUT5 was purified from spermatozoa. Co-immunoprecipitation confirmed the interaction between glycodelin-A and sperm FUT5. Solubilized zona pellucida reduced the binding of glycodelin-A to sperm FUT5. An anti-FUT5 antibody and FUT5 acceptor blocked the binding of glycodelin-A to spermatozoa and the zona binding inhibitory activity of glycodelin-A. Sperm FUT5 bound strongly to intact and solubilized human zona pellucida. The equilibrium dissociation constant of sperm FUT5 binding to solubilized zona pellucida was 42.82 pmol/ml. These observations suggest that human sperm FUT5 is a receptor of glycodelin-A and zona pellucida proteins, and that glycodelin-A inhibits spermatozoa-zona binding by blocking the binding of sperm FUT5 to the zona pellucida.

Zona-Binding Inhibitory Factor-1 from Human Follicular Fluid Is an Isoform of Glycodelin

Abstract Zona-binding inhibitory factor-1 (ZIF-1), a glycoprotein in human follicular fluid, reduces the binding of spermatozoa to the zona pellucida. ZIF-1 has a number of properties similar to those of glycodelin-A from human follicular fluid. The objective of this study was to compare the biochemical characteristics of these two glycoproteins. N-terminal sequencing and protease-digested peptide mapping showed that ZIF-1 and glycodelin-A have the same protein core. However, these glycoproteins differ in their oligosaccharide chains, as demonstrated by fluorophore-assisted carbohydrate electrophoresis, lectin-binding ability, and isoelectric focusing. ZIF-1 inhibited spermatozoa-zona pellucida binding slightly more than did glycodelin-A and significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. Indirect immunofluorescence staining revealed specific binding of glycodelin-A and ZIF-1 to the acrosome region of human spermatozoa, where ZIF-1 produced a stronger signal than did glycodelin-A at the same protein concentration. These data suggest that ZIF-1 is a differentially glycosylated isoform of glycodelin that potently inhibits human sperm-egg interaction. Future study on the function role of ZIF-1 would provide a better understanding of the regulation of fertilization in humans.

Roles of glycodelin in modulating sperm function

Glycodelin is a glycoprotein with three well-defined isoforms. They are named as glycodelin-S, glycodelin-A and glycodelin-F. The three isoforms have similar protein core but different carbohydrate moieties. Glycodelin-S is abundant in the human seminal plasma. It suppresses sperm capacitation and in doing so, it maintains the spermatozoa in an uncapacitated state before they enter into the uterine cavity. Glycodelin-A is abundant in the amniotic fluid. It is also secreted from endometrial glands into uterine fluid and is produced by the fallopian tube. Glycodelin-A is the first endogenous glycoprotein that was found to inhibit the binding of spermatozoa to the zona pellucida. The immunosuppressive properties of glycodelin-A suggest that the molecule may protect the spermatozoa from immune attack in the maternal reproductive tract. Glycodelin-F was first found in the follicular fluid, hence its name. It also inhibits spermatozoa-zona pellucida binding. In addition, glycodelin-F suppresses progesterone-induced acrosome reaction, and may serve to prevent premature acrosome reaction. Preliminary findings suggest possible presence of yet another glycodelin isoform in the extracellular matrix of cumulus oophorus. Unlike glycodelin-A and -F, it stimulates spermatozoa-zona pellucida binding. In summary, different isoforms of glycodelin have different biological roles on sperm function, and they act in succession to contribute to the success of fertilization.

DNA Damage Recognition via Activated ATM and p53 Pathway in Nonproliferating Human Prostate Tissue

DNA damage response (DDR) pathways have been extensively studied in cancer cell lines and mouse models, but little is known about how DNA damage is recognized by different cell types in nonmalignant, slowly replicating human tissues. Here, we assess, using ex vivo cultures of human prostate tissue, DDR caused by cytotoxic drugs (camptothecin, doxorubicin, etoposide, and cisplatin) and ionizing radiation (IR) in the context of normal tissue architecture. Using specific markers for basal and luminal epithelial cells, we determine and quantify cell compartment-specific damage recognition. IR, doxorubicin, and etoposide induced the phosphorylation of H2A.X on Ser(139) (γH2AX) and DNA damage foci formation. Surprisingly, luminal epithelial cells lack the prominent γH2AX response after IR when compared with basal cells, although ATM phosphorylation on Ser(1981) and 53BP1 foci were clearly detectable in both cell types. The attenuated γH2AX response seems to result from low levels of total H2A.X in the luminal cells. Marked increase in p53, a downstream target of the activated ATM pathway, was detected only in response to camptothecin and doxorubicin. These findings emphasize the diversity of pathways activated by DNA damage in slowly replicating tissues and reveal an unexpected deviation in the prostate luminal compartment that may be relevant in prostate tumorigenesis. Detailed mapping of tissue and cell type differences in DDR will provide an outlook of relevant responses to therapeutic strategies.

Cumulus Oophorus-associated Glycodelin-C Displaces Sperm-bound Glycodelin-A and -F and Stimulates Spermatozoa-Zona Pellucida Binding

Spermatozoa have to swim through the oviduct and the cumulus oophorus before fertilization in vivo. In the oviduct, spermatozoa are exposed to glycodelin-A and -F that inhibit spermatozoa-zona pellucida binding. In this study, we determined whether these glycodelins would inhibit fertilization. The data showed that the spermatozoa without previous exposure to glycodelin-A and -F acquired glycodelin immunoreactivity during their passage through the cumulus oophorus. On the other hand, when glycodelin-A or -F-pretreated spermatozoa were exposed to the cumulus oophorus, the zona pellucida binding inhibitory activity of glycodelin-A and -F was not only removed, but the spermatozoa acquired enhanced zona pellucida binding ability. These actions of the cumulus oophorus were due to the presence of a cumulus isoform of glycodelin, designated as glycodelin-C. The cumulus cells could convert exogenous glycodelin-A and -F to glycodelin-C, which was then released into the surrounding medium. The protein core of glycodelin-C was identical to that in other glycodelin isoforms, as demonstrated by mass spectrum, peptide mapping, and affinity to anti-glycodelin antibody recognizing the protein core of glycodelin. In addition to having a smaller size and a higher isoelectric point, glycodelin-C also had lectin binding properties different from other isoforms. Glycodelin-C stimulated spermatozoazona pellucida binding in a dose-dependent manner, and it effectively displaced sperm-bound glycodelin-A and -F. In conclusion, the cumulus cells transform glycodelin-A and -F to glycodelin-C, which in turn removes the spermatozoazona binding inhibitory glycodelin isoforms and enhances the zona binding capacity of spermatozoa passing through the cumulus oophorus.

Glycodelin and β‐lactoglobulin, lipocalins with a high structural similarity, differ in ligand binding properties

Human glycodelin, a lipocalin with a high amino acid similarity to β‐lactoglobulins, appears as various glycoforms with different biological activities in endometrium (glycodelin‐A) and seminal plasma (glycodelin‐S). We found that the structures of these glycodelins and β‐lactoglobulin are similar. Despite this structural similarity, unlike β‐lactoglobulin, glycodelin‐A binds neither retinoic acid nor retinol. It was impossible to detect any endogenous retinoids or steroids in any of the two purified glycodelins. Both their glycoforms share similar thermodynamic parameters of reversible denaturation suggesting that native folding of glycodelin‐A and glycodelin‐S is not influenced by the differences in glycosylation or by ligand binding.

Effects of differential glycosylation of glycodelins on lymphocyte survival.

Glycodelin is a human glycoprotein with four reported glycoforms, namely glycodelin-A (GdA), glycodelin-F (GdF), glycodelin-C (GdC), and glycodelin-S (GdS). These glycoforms have the same protein core and appear to differ in their N-glycosylation. The glycosylation of GdA is completely different from that of GdS. GdA inhibits proliferation and induces cell death of T cells. However, the glycosylation and immunomodulating activities of GdF and GdC are not known. This study aimed to use ultra-high sensitivity mass spectrometry to compare the glycomes of GdA, GdC, and GdF and to study the relationship between the immunological activity and glycosylation pattern among glycodelin glycoforms. Using MALDI-TOF strategies, the glycoforms were shown to contain an enormous diversity of bi-, tri-, and tetra-antennary complex-type glycans carrying Galβ1–4GlcNAc (lacNAc) and/or GalNAcβ1–4GlcNAc (lacdiNAc) antennae backbones with varying levels of fucose and sialic acid substitution. Interestingly, they all carried a family of Sda (NeuAcα2–3(GalNAcβ1–4)Gal)-containing glycans, which were not identified in the earlier study because of less sensitive methodologies used. Among the three glycodelins, GdA is the most heavily sialylated. Virtually all the sialic acid on GdC is located on the Sda antennae. With the exception of the Sda epitope, the GdC N-glycome appears to be the asialylated counterpart of the GdA/GdF glycomes. Sialidase activity, which may be responsible for transforming GdA/GdF to GdC, was detected in cumulus cells. Both GdA and GdF inhibited the proliferation, induced cell death, and suppressed interleukin-2 secretion of Jurkat cells and peripheral blood mononuclear cells. In contrast, no immunosuppressive effect was observed for GdS and GdC.