Differences between neonates and adults in tissue-type-plasminogen activator (t-PA)-catalyzed plasminogen activation with various effectors and in carbohydrate sequences of fibrinogen chains.

Our study investigates the effect of fetal and adult soluble fibrin (SF), fetal and adult fibrinogen Aalpha- and gamma-chains, as well as adult CNBr-fibrinogen fragments on tissue-type plasminogen activator (t-PA)-catalyzed plasminogen activation of both fetal and adult Glu-plasminogen types 1 and 2. In addition, we determined carbohydrate sequences of fetal and adult Bbeta- and gamma-chains by mass spectrometric analysis. In the absence of an effector, no substantial differences in the rate of plasmin formation could be seen between the fetal and adult plasminogen types. In the presence of an effector, both fetal Glu-plasminogen types revealed lower values for k(cat app) than the respective adult types. No differences could be seen in the values for K(m app). The resulting differences in catalytic efficiencies between the fetal and adult plasminogen types were much less than previously reported. No differences could be seen between fetal and adult effectors in stimulating t-PA-catalyzed plasminogen activation. Detailed analyses of the activation kinetics revealed a longer initial phase of slow plasmin formation of both fetal Glu-plasminogen types compared to their respective adult types, indicating a slower plasmin-induced modification of CNBr-fibrinogen fragments or SF by fetal plasmin. Mass spectrometric analysis of the N-glycans present on adult and fetal Bbeta- and gamma-fibrinogen chains showed the presence of a major monosialylated biantennary structure with lesser amounts of the disialylated form. In contrast to previous data, we conclude that catalytic efficiency of t-PA-catalyzed plasminogen activation in neonates is only slightly lower than in adults.

Galactofuranoic-oligomannose N-linked glycans of alpha-galactosidase A from Aspergillus niger.

Extracellular alpha-galactosidase A was purified from the culture filtrate of an over-producing strain of Aspergillus niger containing multiple copies of the encoding aglA gene under the control of the glucoamylase (glaA) promoter. Endoglycosidase digestion followed by SDS/PAGE, lectin and immunoblotting suggested that glycosylation accounted for approximately 25% of the molecular size of the purified protein. Monosaccharide analysis showed that this was composed of N-acetyl glucosamine, mannose and galactose. Mild acid hydrolysis, mild methanolysis, immunoblotting and exoglycosidase digestion indicated that the majority of the galactosyl component was in the furanoic conformation (beta-D-galactofuranose, Galf). At least 20 different N-linked oligosaccharides were fractionated by high-pH anion-exchange chromatography following release from the polypeptide by peptide-N-glycosidase F. The structures of these were subsequently determined by fast atom bombardment mass spectrometry to be a linear series of Hex(7-26)HexHA(c2). Indicating that oligosaccharides from GlcNA(c2)Man(7), increasing in molecular size up to GlcNA(c2)Man(24) were present. Each of these were additionally substituted with up to three beta-Galf residues. Linkage analysis confirmed the presence of mild acid labile terminal hexofuranose residues. These results show that filamentous fungi are capable of producing a heterogeneous mixture of high molecular-size N-linked glycans substituted with galactofuranoic residues, on a secreted glycoprotein.

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.

Recombinant glycodelin carrying the same type of glycan structures as contraceptive glycodelin-A can be produced in human kidney 293 cells but not in chinese hamster ovary cells.

We have produced human recombinant glycodelin in human kidney 293 cells and in Chinese hamster ovary (CHO) cells. Structural analyses by lectin immunoassays and fast atom bombardment mass spectrometry showed that recombinant human glycodelin produced in CHO cells contains only typical CHO-type glycans and is devoid of any of the N, N'-diacetyllactosediamine (lacdiNAc)-based chains previously identified in glycodelin-A (GdA). By contrast, human kidney 293 cells produced recombinant glycodelin with the same type of carbohydrate structures as GdA. The presence of a beta1-->4-N-acetylgalactosaminyltransferase functioning in the synthesis of lacdiNAc-based glycans in human kidney 293 cells is concluded to be the cause of the occurrence of lacdiNAc-based glycans on glycodelin produced in these cells. Furthermore, human kidney 293 cells were found to be particularly suited for the production of recombinant glycodelin when they were cultured in high glucose media. Lowering the glucose concentration and the addition of glucosamine resulted in higher relative amounts of oligomannosidic-type glycans and complex glycans with truncated antennae. Human glycodelin is an attractive candidate for the development of a contraceptive agent, and this study gives valuable information for selecting the proper expression system and cell culture conditions for the production of a correctly glycosylated recombinant form.

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.

Modification of a recombinant GPI-anchored metalloproteinase for secretion alters the protein glycosylation.

The N-linked glycans of recombinant leishmanolysin (GP63) expressed as a glycosylphosphatidylinositol (GPI)-anchored membrane protein or modified for secretion in Chinese hamster ovary (CHO) cells were analyzed by fast atom bombardment-mass spectrometry (FAB-MS). The glycans isolated from both membrane and secreted protein were predominantly complex biantennary structures. However other aspects of the glycan profiles showed striking differences. The degree of sialylation of the membrane form was greatly reduced and the core fucosylation of biantennary structures was increased compared to the secreted form. Glycans isolated from membrane expressed protein also contained a higher proportion of lactosamine repeats. Residence times in the secretory pathway were similar for both secreted and membrane protein. Glycosylation differences may therefore be due to differences in protein conformation and accessibility to glycosyltransferases or glycosidases. These differences in glycosylation represent an important factor when considering modifying membrane expressed proteins for secreted production.

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.

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.

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.

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.

Two-stage spatial filtering for diffraction pattern analysis.

The use of optical power spectrum analysis for feature detection and classification is often restricted by the superposition of the aperture spectrum. A spatial frequency filtering technique using a circular aperture and filters has been previously proposed to separate the object and aperture spectra. An adaptation of this technique using square apertures and an opaque cross spatial filter offers improved performance for some applications. Numerical calculations and experimental results are given comparing the two techniques.