Characterization of monoclonal antibodies to glycodelin and recombinant glycodelin

Glycodelin-A belongs to the lipocalin superfamily. Although it is associated with normal endometrial growth during the menstrual cycle, fertilization and normal pregnancy in humans, the molecular mechanism of its biological action has not been elucidated. To undertake studies to understand the functional relevance of any molecule, obtaining large quantities of the protein becomes essential. With the ultimate aim of purifying glycodelin either from its natural sources (human amniotic fluid) or the recombinant glycodelin from bacterial recombinant lysates, we raised monoclonal antibodies to this protein. As immunogens, recombinant glycodelin expressed in E. coli and Pichia pastoris as well as glycodelin from amniotic fluid were used. The monoclonal antibodies generated were characterized with respect to binding to both the native as well as the recombinant proteins using ELISA, immunoblotting, and immunohistochemistry.     

Oxidants and antioxidants affect the expression of glycodelin

Glycodelin is a glycoprotein that has immunosuppressive activity. We have shown that K562 cells, hematopoitic progenitor cells, are capable of synthesizing glycodelin peptide (Gp) and, perhaps, contribute to Gp in tissues. In addition, several reproductive and nonreproductive tissues themselves are capable of synthesis of glycodelin. In this study, we report that lipid peroxides induce the synthesis of Gp. Antioxidants vitamin E and pyrrolidine dithiocarbamate (PDTC) and antioxidizing enzymes catalase and superoxide dismutase (SOD) effectively blocked phorbol myristate acetate- (PMA-) and lyso phosphatidic acid- (LPA-) induced synthesis of Gp. Dioctanoin (a mimic of diacylglycerol) activated Gp synthesis, and an inhibitor of protein kinase C (PKC) downregulated the response. Based on these observations, we postulate that oxidants by way of PKC might potentiate the angiogenic process.     

Oligosaccharides modulate the apoptotic activity of glycodelin

GlycodelinA (GdA), a multifunctional glycoprotein secreted at high concentrations by the uterine endometrium during the early phases of pregnancy, carries glycan chains on asparagines at positions N28 and N63. GdA purified from amniotic fluid is known to be a suppressor of T-cell proliferation, an inducer of T-cell apoptosis, and an inhibitor of sperm-zona binding in contrast to its glycoform, glycodelinS (GdS), which is secreted by the seminal vesicles into the seminal plasma. The oligosaccharide chains of GdA terminate in sialic acid residues, whereas those of GdS are not sialylated but are heavily fucosylated. Our previous work has shown that the apoptogenic activity of GdA resides in the protein backbone, and we have also demonstrated the importance of sialylation for the manifestation of GdA-induced apoptosis. Recombinant glycodelin (Gd) expressed in the Sf21 insect cell line yielded an apoptotically active Gd; however, the same gene expressed in the insect cell line Tni produced apoptotically inactive Gd, as observed with the gene expressed in the Chinese hamster ovary (CHO) cell line and earlier in Pichia pastoris. Glycan analysis of the Tni and Sf21 cell line-expressed Gd proteins reveals differences in their glycan structures, which modulate the manifestation of apoptogenic activity of Gd. Through apoptotic assays carried out with the wild-type (WT) and glycosylation mutants of Gd expressed in Sf21 and Tni cells before and after mannosidase digestion, we conclude that the accessibility to the apoptogenic region of Gd is influenced by the size of the glycans.     

Mate‐sampling costs and sexy sons

Costly female mating preferences for purely Fisherian male traits (i.e. sexual ornaments that are genetically uncorrelated with inherent viability) are not expected to persist at equilibrium. The indirect benefit of producing ‘sexy sons’ (Fisher process) disappears: in some models, the male trait becomes fixed; in others, a range of male trait values persist, but a larger trait confers no net fitness advantage because it lowers survival. Insufficient indirect selection to counter the direct cost of producing fewer offspring means that preferences are lost. The only well‐cited exception assumes biased mutation on male traits. The above findings generally assume constant direct selection against female preferences (i.e. fixed costs). We show that if mate‐sampling costs are instead derived based on an explicit account of how females acquire mates, an initially costly mating preference can coevolve with a male trait so that both persist in the presence or absence of biased mutation. Our models predict that empirically detecting selection at equilibrium will be difficult, even if selection was responsible for the location of the current equilibrium. In general, it appears useful to integrate mate sampling theory with models of genetic consequences of mating preferences: being explicit about the process by which individuals select mates can alter equilibria.     

Differential sialylation regulates the apoptotic activity of glycodelin A

Glycodelin A (GdA), a dimeric lipocalin, expressed by the uterine endometrium, is an immunomodulatory agent and induces apoptosis in T-cells. In this study we demonstrate that two populations of GdA with subtle differences in their net ionic charge are present in the amniotic fluid and that, apoptotic activity is exhibited only by the population with more sialic acid residues. Significantly, removal of sialic acid residues from the active populations of GdA abrogates the activity of the molecule, suggesting that the extent of sialylation might be a factor regulating the activity of GdA.     

Immunolocalization of glycodelin in the genital tract of rats

Glycodelin, also known as placental protein 14 has been predominantly localized to organs of the human genital tract. Unfortunately the physiological role of glycodelin is largely unknown since it depends on limited availability of tissues. Therefore, a suitable animal model to study the role of glycodelin would be desirable. Previously, it was shown that glycodelin mRNA is expressed in the genital tract of male and female rats. In the present study, we demonstrate the expression of glycodelin protein in male and female rats by immunohistochemistry and Western blot analysis. For this purpose a polyclonal antibody was generated against glycodelin peptide. In female rats, glycodelin was found in the epithelial gland cells of the uterus, epithelial cells of the fallopian tube as well as in corpora lutea, interstitial and theca cells of the ovary. Glycodelin was distributed in all epithelial cells of the epididymis and the seminal vesicle. In the seminiferous epithelium, glycodelin was seen in all developmental stages of spermatogonia and spermatocytes and in Sertoli cells. Whereas in the rat male reproductive tract glycodelin expression is slightly different from human or primate tissues, in organs of the rat female genital tract glycodelin expression is similar to humans and primates.     

Purification and characterization of an immunomodulatory endometrial protein, glycodelin

Human glycodelin is synthesized by endometrial cells in the late secretory phase and early pregnancy under hormonal regulation. Whereas the precise physiological functions of glycodelin are unknown, its expression during embryonic nidation and its inhibition of T cell proliferation suggest an immunomodulatory role. We purified human glycodelin from first trimester human decidual cytosol by using a rapid two-step high-performance liquid chromatography method and investigated its effects on human monocyte migration. Human U937 cells were used as a model of monocyte chemotaxis in Boyden chamber migration assays. N-Formyl-Met-Leu-Phe and the beta-chemokine RANTES (regulated on activation normal T cell expressed and secreted) were used as monocyte chemoattractants. Purified glycodelin inhibited monocyte migration in a dose-dependent fashion (IC50 = 550 nm). Glycodelin activity was totally reversed by heat inactivation (95 degrees C x 15 min) and neutralized by pretreatment with specific anti-glycodelin antibodies. Deglycosylated glycodelin was equipotent to intact glycodelin in the monocyte migration assay. 125I-Glycodelin binding to whole U937 cells revealed a single, saturable site with a Kd = 48 +/- 21 nm by Scatchard analysis. Cross-linking studies indicated that glycodelin binds to a high molecular mass (approximately 250 kDa) protein complex at the monocyte cell surface. Our findings support the hypothesis that glycodelin reduces the local maternal inflammatory response toward the implantation of a semiallogeneic conceptus.     

Differences in glycosylation and sperm-egg binding inhibition of pregnancy-related glycodelin

Glycodelin is a glycoprotein produced in many glands, particularly those of reproductive tissues. It appears as different glycoforms in amniotic fluid (glycodelin-A) and seminal plasma (glycodelin-S), but only glycodelin-A inhibits gamete adhesion. In the present study, glycodelin from secretory-phase endometrium, first-trimester pregnancy decidua, and midtrimester amniotic fluid was studied with respect to physicochemical properties, including glycosylation patterns and inhibitory activity of sperm-egg binding. Purified glycodelins from all these sources were similar in isoelectric focusing and in lectin immunoassays using lectins from Wisteria floribunda and Sambucus nigra. Likewise, the glycodelins inhibited sperm-egg binding in a dose-dependent manner, as measured by hemizona-binding assay. However, subtle quantitative physicochemical and biological differences were found between glycodelins from different sources as well as within the same tissue/fluid between different individuals. Differences were most pronounced between endometrial glycodelins from nonpregnancy and first-trimester pregnancy. The glycan structures studied by fast-atom bombardment mass spectrometry of individual amniotic fluid glycodelin-A samples also showed interindividual quantitative differences. In conclusion, glycodelins from different female reproductive tract tissues and amniotic fluid share substantial similarity, allowing all of them to be called glycodelin-A. However, these glycodelins exhibit quantitative physicochemical and functional differences between different sources and individuals.     

Effects of ammonia and glucosamine on the heterogeneity of erythropoietin glycoforms

Recombinant human erythropoietin (EPO) is a glycoprotein produced as a therapeutic agent from mammalian cell cultures for the treatment of anemia associated with severe kidney damage. The EPO structure has a high glycan content which is essential for bioactivity but shows considerable molecular heterogeneity. The cell culture conditions that affect the heterogeneity of the glycoforms of EPO are not well understood. However, the accumulation of ammonia in culture is one factor that has been associated with an enhanced heterogeneity of glycoforms. In this report we investigate the metabolic perturbations associated with ammonia and glucosamine that may give rise to an altered pattern of EPO glycosylation. Recombinant human erythropoietin was synthesized in serum-free cultures of transfected Chinese hamster ovary (CHO) cells. The molecular heterogeneity of erythropoietin was increased by supplementation of cultures with either ammonia or glucosamine. The enhanced molecular heterogeneity was shown to be due to variable glycosylation that resulted in EPO with an enhanced molecular weight and isoelectric point range. Enzymatic removal of the glycan moiety of EPO in all cases resulted in a single molecular form with a molecular weight of 18 000, which corresponded to non-glycosylated EPO. The variable glycosylation was consistent with reduced sialylation and antennarity of the carbohydrate structures present on the three N-linked sites of EPO. In the presence of ammonia (>30 mM) the proportion of tetrasialylated and tetraantennary glycan structures were reduced by 73% and 57%, respectively, as determined by HPLC analysis. Such changes were also observed, although to a lesser extent (41% and 37%), by an increase in the glucosamine concentration (>10 mM) in the medium. The enhanced heterogeneity of the glycan structures coincided with a significant increase in the intracellular UDP-N-acetylhexosamine (UDP-GNAc) pool. The measured UDP-GNAc level was up to 2 orders of magnitude higher in the presence of either glucosamine or ammonia. However, the changes in the glycosylation profiles induced by either glucosamine or ammonia were significantly different even at the same intracellular UDP-GNAc concentration. This suggests that the enhanced EPO heterogeneity could not be mediated solely by the increased UDP-GNAc level. Glucosamine (but not ammonia) was shown to cause significant inhibition of glucose transport into the cells, which could induce a different pattern of primary metabolism.