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G F Clark

Publications and source records attributed to G F Clark.

At least 19 recordsLinked to original sources
PubMed2001

The species recognition system: a new corollary for the human fetoembryonic defense system hypothesis.

We have previously suggested that the human fetus is protected during human development by a system of both soluble and cell surface associated glycoconjugates that utilize their carbohydrate sequences as functional groups to enable them to evoke tolerance. The proposed model has been referred to as the human fetoembryonic defense system hypothesis (hu-FEDS). In this paradigm, it has previously been proposed that similar oligosaccharides are used to mediate crucial recognition events required during both human sperm-egg binding and immune-inflammatory cell interactions. This vertical integration suggested to us that the sperm-egg binding itself is related to universal recognition events that occur between immune and inflammatory cells, except that in this case recognition of 'species' rather than recognition of 'self' is being manifested. In this paper, we have designated this component of hu-FEDS as the species recognition system (SRS). We propose that the SRS is an integral component of the hu-FEDS used to enable sperm-egg recognition and protection of the gametes from potential immune responses. Recent structural data indicates that the glycan sequences implicated in mediating murine gamete recognition are also expressed on CD45 in activated murine T lymphocytes and cytotoxic T lymphocytes. This overlap supports our contention that there is an overlap between the immune and gamete recognition systems. Therefore the hu-FEDS paradigm may be a subset of a larger model that also applies to other placental mammals. We therefore propose that the hu-FEDS model for protection should in the future be referred to as the eutherian fetoembryonic defense system hypothesis (eu-FEDS) to account for this extension. The possibility exists that the SRS component of eu-FEDS could predate eutherians and extend to all sexually reproducing organisms. Future investigation of the interactions between the immune and gamete recognition system will be required to determine the degree of overlap.

Embryo, Mammalian↗
PubMed2000

Pregnancy-associated changes in the glycosylation of tamm-horsfall glycoprotein. Expression of sialyl Lewis(x) sequences on core 2 type O-glycans derived from uromodulin.

Tamm-Horsfall glycoprotein (THP) is a major glycoprotein associated with human urine that binds pro-inflammatory cytokines and also inhibits in vitro T cell proliferation induced by specific antigens. THP derived from human pregnancy urine (designated uromodulin) has previously been shown to be 13-fold more effective as an inhibitor of antigen-induced T cell proliferation than THP obtained from other sources. Structural analysis of human THP and uromodulin has for the first time revealed that these glycoproteins are O-glycosylated. THP from nonpregnant females and males expresses primarily core 1 type O-glycans terminated with either sialic acid or fucose but not the sialyl Lewis(x) epitope. By contrast, the O-glycans linked to uromodulin include unusual core 2 type glycans terminated with one, two, or three sialyl Lewis(x) sequences. The specific association of these unusual carbohydrate sequences with uromodulin could explain its enhanced immunomodulatory effects compared with THP obtained from males and nonpregnant females. Analysis of THP from one of the pregnant females 2 months postpartum showed a reversion of the O-glycan profile to that found for a non-pregnant female. These data suggest that the glycosylation state of uromodulin could be under the regulation of steroidal hormones produced during pregnancy. The significant physiological implications of these observations are discussed.

Female↗
PubMed2000

Structural analysis of murine zona pellucida glycans. Evidence for the expression of core 2-type O-glycans and the Sd(a) antigen.

Murine sperm initiate fertilization by binding to specific oligosaccharides linked to the zona pellucida, the specialized matrix coating the egg. Biophysical analyses have revealed the presence of both high mannose and complex-type N-glycans in murine zona pellucida. The predominant high mannose-type glycan had the composition Man(5)GlcNAc(2), but larger oligosaccharides of this type were also detected. Biantennary, triantennary, and tetraantennary complex-type N-glycans were found to be terminated with the following antennae: Galbeta1-4GlcNAc, NeuAcalpha2-3Galbeta1-4GlcNAc, NeuGcalpha2-3Galbeta1-4GlcNAc, the Sd(a) antigen (NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc, NeuGcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc), and terminal GlcNAc. Polylactosamine-type sequence was also detected on a subset of the antennae. Analysis of the O-glycans indicated that the majority were core 2-type (Galbeta1-4GlcNAcbeta1-6[Galbeta1-3]GalNAc). The beta1-6-linked branches attached to these O-glycans were terminated with the same sequences as the N-glycans, except for terminal GlcNAc. Glycans bearing Galbeta1-4GlcNAcbeta1-6 branches have previously been suggested to mediate initial murine gamete binding. Oligosaccharides terminated with GalNAcbeta1-4Gal have been implicated in the secondary binding interaction that occurs following the acrosome reaction. The significant implications of these observations are discussed.

Animals↗
PubMed1998

Glycodelins: role in regulation of reproduction, potential for contraceptive development and diagnosis of male infertility.

Glycodelins are glycoproteins synthesized in various glands, with sequence homology to beta-lactoglobulins, and named according to their unique oligosaccharide structures. We purified, cloned and sequenced endometrium- and seminal plasma-derived glycodelins (GdA and GdS respectively) and found that they are involved in various types of cell-cell communications. These include interactions between the spermatozoon and the egg, and between immune cells and their targets. Endometrial GdA inhibits sperm-egg binding, whereas the differently glycosylated GdS in seminal plasma does not. These observation are of interest for reproductive physiology, detection of causes of infertility, and they also may have potential for contraceptive development.

Biomarkers↗
PubMed1998

Direct evidence for the involvement of carbohydrate sequences in human sperm-zona pellucida binding.

Several lines of evidence indicate that mammalian fertilization is initiated via a binding process that is dependent upon the recognition of oligosaccharide sequences associated with zona pellucida (ZP) glycoproteins. Here, specific chemical and enzymatic methods were employed to modify human ZP and to test their effects on sperm binding in the hemizona assay system (HZA). Periodate oxidation of human ZP under very mild conditions (10 min, 0 degrees C, 1 mM sodium m-periodate) that attacks only terminal sialic acid resulted in a 30% loss of human sperm binding in the HZA [hemizona index (HZI) = 70.2 +/- 10.9, n = 22; P < 0.05]. Periodate oxidation under mild conditions (1 h, 23 degrees C, 10 mM sodium m-periodate) caused a 40% decrease in binding (HZI = 60.8 +/- 10.3; n = 24; P< 0.01). Treatment of human ZP with neuraminidase caused a substantial increase in sperm binding to human ZP (HZI = 297 +/- 45, n = 22; P < 0.01). These findings indicate that there are sialic acid dependent binding sites coexisting with binding sites that are obscured by sialic acid. To determine the periodate sensitivity of these obscured sites, hemizona were first digested with neuraminidase and subsequently subjected to mild periodate oxidation. The combined enzymatic and chemical treatments caused a 79% decrease in sperm binding compared to control hemizona (HZI = 20.7 +/- 4.4, n = 16; P < 0.001). Human sperm-ZP interaction was also increased by digestion of human ZP with endo-beta-galactosidase (HZI = 710 +/- 232, n = 14; P < 0.01), indicating that potential binding sites for spermatozoa are also obscured by lactosaminoglycan sequences. These studies support a definitive role for the involvement of ZP-associated glycans in the binding of human spermatozoa to oocytes.

Carbohydrate Sequence↗
PubMed1997

The malaria circumsporozoite protein: interaction of the conserved regions I and II-plus with heparin-like oligosaccharides in heparan sulfate.

The malaria circumsporozoite (CS) protein binds to glycosaminoglycans from heparan sulfate proteoglycans on the cell surface of hepatocytes and is specifically cleared from the bloodstream by the liver. We show here that the two conserved regions, I and II-plus, of the CS protein, in a concerted action, preferentially bind to highly sulfated heparin-like oligosaccharides in heparan sulfate. In a concentration-dependent manner, peptides representing region I and region II-plus inhibited the binding of recombinant CS protein to HepG2 cells by 62 and 84%, respectively. Furthermore, the action of endoproteinase Arg-C, which cleaves the recombinant CS constructs CS27IVC and CSFZ(Cys) predominantly at the conserved region I, was inhibited by heparin in a concentration-dependent fashion. CSFZ(Cys), which has a higher affinity to HSPGs than CS27IVC, was stabilized by heparin at a w/w ratio (CS protein:glycosaminoglycan) of 20/1, whereas full protection of CS27IVC required more heparin (5/1). Heparan sulfate provided full protection of CSFZ(Cys) only at a ratio of 1/10. Native fucoidan as well as normally sulfated fuco-oligosaccharides (0.76 mol sulfate/mol fucose) inhibited Plasmodium berghei development in HepG2 cells by 84 and 66%, respectively, in a concentration-dependent manner and sporozoite invasion into CHO cells by 80%. Desulfated fucoidan oligosaccharides were inactive. These results may explain the selective interaction between the CS protein and the unique heparan sulfate from liver, which is noted for its unusually high degree of sulfation, and may provide a plausible explanation for the selective targeting of the malaria CS protein to the liver.

Amino Acid Sequence↗
PubMed1997

Viewing AIDS from a glycobiological perspective: potential linkages to the human fetoembryonic defence system hypothesis.

The primary molecular changes that lead to development of acquired immunodeficiency syndrome (AIDS) are very poorly understood, as are the mechanisms underlying the protection of the developing human from the maternal immune response. Recent data that the human immunodeficiency virus (HIV) may be using the glycosylation system of the T lymphocytes to acquire glycans for its glycoproteins that enable it to disrupt carbohydrate dependent immune cell interactions or induce aberrant immune reactions. Consistent with this hypothesis, gp120 from HIV infected human H9 lymphoblastoid cells expresses biantennary N-linked glycans with a bisecting GlcNAc sequence on 11% of their total oligosaccharides. This specific carbohydrate sequence has recently been shown to protect K562 erythroleukemic cells from natural killer (NK) cell responses when presented on the cell surface. We have recently demonstrated that bisecting biantennary type N-linked glycans are also expressed on the human zona pellucida (ZP); previous lectin binding studies indicate that is also expressed on human spermatozoa. Thus both the human gametes and HIV produced by H9 cells carry this same protective carbohydrate epitope on their outer surfaces. Human alpha-fetoprotein expressed in the developing human also carries the bisecting GlcNAc sequence, indicating that it may be suppressing the emerging fetal immune response by using its carbohydrate sequence as a functional group. We have suggested that the developing human and the gametes are also protected by soluble immunosuppressive glycoproteins found in the amniotic fluid and seminal plasma known as glycodelin-A (GdA) and glycodelin-S (GdS) respectively. Structural analysis of their N-linked oligosaccharides combined with other functional studies suggest that GdA and GdS employ their very unusual carbohydrate sequences as functional groups that enable them to manifest their immunosuppressive activities. GdA and GdS are significant components of our recently proposed model for the protection of the developing human and gametes designated the human fetoembryonic defence system hypothesis. A striking relationship now emerging is that the same unusual carbohydrate sequences associated with these immunosuppressive glycodelins are also specifically expressed on intravascular helminthic parasites, Helicobacter pylori, human tumour cells, and HIV infected T lymphocytes. The information presented in this review suggests that two new corollaries should be added to our recently proposed defence system hypothesis: (i) mimicry or acquisition of glycans that are used in this protective system by pathogens or tumour cells may enable them to either subvert or misdirect the human immune response, thereby greatly increasing their pathogenicity; and (ii) expression of glycoproteins used in this system by normal cells and tissues outside the reproductive system may protect them from immune responses, especially in those cases where major histocompatibility recognition is either absent or minimal. A better understanding of this hypothesis and its corollaries may enable us to address the molecular mechanisms underlying not only AIDS but also a host of other very serious pathological conditions in the human.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Expression of glycans linked to natural killer cell inhibition on the human zona pellucida.

Protection of the gametes from potential immune responses is a primary function in human reproduction. The primary cell type responsible for the innate immune response in the uterus is the natural killer (NK) cell. NK cells normally recognize Class I major histocompatibility (MHC) molecules on potential target cells. Since both human spermatozoa and human oocytes do not express Class I MHC molecules on their surfaces, the appropriate cell surface signal that abrogates potential NK cell-mediated responses directed against these gametes is unknown. Recent evidence indicates that surface expression of bisecting-type N-linked glycans protects cells sensitive to NK cell-mediated lysis. We report that the zona pellucida of the human egg and plasma membranes of human spermatozoa potentially bind a lectin probe specific for bisecting type glycans in a carbohydrate-dependent manner. Since the innate immune response in the uterus is primarily mediated by NK cells, our results indicate that human gametes may be protected from this response by expressing bisecting type N-linked glycans on their surfaces.

Carbohydrate Conformation↗
PubMed1996

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 (GalNAcbeta1-4GlcNAc) 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 (Galbeta1-4(Fucalpha1-3)GlcNAc) and Lewisy (Fucalpha1-2Galbeta1-4(Fucalpha1-3)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.

Contraceptive Agents↗
PubMed1996

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.

Animals↗
PubMed1996

Glycodelin from seminal plasma is a differentially glycosylated form of contraceptive glycodelin-A.

Glycodelin-A is a human amniotic fluid-derived glycoprotein with contraceptive and immunosuppressive activities. An immunoreactive form of glycodelin was detected in seminal plasma over a decade ago, but definitive characterization of this glycoprotein was not pursued. We considered it unlikely that the seminal plasma of fertile men would contain an appreciable amount of contraceptive glycodelin-A. To address this issue we purified seminal plasma glycodelin (glycodelin-S) and performed comparative studies with glycodelin-A. Glycodelin-S behaved differently when compared with glycodelin-A during sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing but identically after enzymatic deglycosylation. N-terminal sequencing of glycodelin-A and glycodelin-S gave identical results, and digestion with trypsin gave identical peptide fragments. The glycoproteins were also found to be indistinguishable from each other based upon immunological analyses. These results indicate that glycodelin-S and glycodelin-A have similar overall protein structure, suggesting the likelihood that these glycoproteins are differentially glycosylated forms of very similar proteins. This latter possibility is supported by lectin binding studies indicating that, unlike glycodelin-A, glycodelin-S does not manifest any affinity for lectins from Wisteria floribunda or Sambucus nigra. The results of sugar analysis and neuraminidase digestion also lead us to conclude that glycodelin-S and glycodelin-A are differentially glycosylated forms of similar proteins. Our evidence indicates that glycodelin-A mediated its biological activities via its unusual oligosaccharide sequences that are not associated with glycodelin-S. In lectin-immunoassay no appreciable amount of contraceptive glycodelin-A was found in the 22 seminal plasma samples studied.

Amidohydrolases↗
PubMed1996

Providing effective occupational therapy services: data-based decision making in school-based practice.

Educational practice is moving away from an "eligibility-oriented" student-problem approach toward a problem-solving approach that connects evaluation and interventions. This approach involves the identification of variables used to frame interventions, and, ultimately, it permits evaluation of intervention effectiveness. This article describes a problem-solving model developed and used within the Heartland Area Education Agency. This model relies on collecting baseline data and ongoing data, producing a basis not only for decision making, but also for evaluating the chosen intervention. This systematic and collaborative approach is used by the educational team to identify concerns about students' academic and nonacademic performance, to plan interventions, and to establish measurable outcomes.

Data Collection↗
PubMed1995

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: Gal beta 1-4GlcNAc (lacNAc), GalNAc beta 1-4GlcNAc (lacdiNAc), NeuAc alpha 2-6Gal beta 1-4GlcNAc (sialylated lacNAc), NeuAc alpha 2-6Gal beta 1-4GlcNAc (sialylated lacdiNAc), Gal beta 1-4(Fuc alpha 1-3)GlcNAc (Lewisx), and GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc (lacdiNAc analogue of Lewisx). 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.

Amino Acid Sequence↗
PubMed1995

New concepts in human sperm-zona pellucida interaction.

Binding of spermatozoa to the zona pellucida is an initial, crucial recognition event leading to fertilization. In the mouse, the best species characterized so far, the zona pellucida protein 3 (ZP3) has a central role as the specific, primary sperm receptor on the zona and as the inducer of the acrosome reaction. This sequence of events is not clearly understood as it relates to human gametes. The ideal test for the evaluation of sperm-zona pellucida interaction is one that can examine these events in a sequential fashion (i.e. binding followed by the acrosome reaction) in a standardized and specific bioassay. Here we have used the hemizona assay as an internally controlled test to examine human sperm-zona pellucida interaction. Results presented show that: (i) the hemizona assay has an excellent predictive power for IVF outcome and for the identification of male infertility; (ii) using a specific anti-ZP3 antiserum in immunocytochemical studies, the hemizona assay allows for the identification of structural/functional anomalies of the protein backbone of human ZP3 (identification of oocyte anomalies); and (iii) glycobiological studies using the hemizona assay model indicate that the initial sperm-zona pellucida binding requires a seletin-like interaction between the human gametes. These efforts may help us to characterize the cellular and molecular mechanisms involved in human gametes and their dysfunctions in infertile patients.

Animals↗