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J. Biol. Chem., Vol. 277, Issue 36, 32562-32570, September 6, 2002

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The Expression of Free Oligosaccharides in Human Seminal Plasma^*

Sara Chalabi, Richard L. Easton, Manish S. Patankar^§, Frank A. Lattanzio^§, Jamie C. Morrison^§, Maria Panico, Howard R. Morris^¶, Anne Dell, and Gary F. Clark^§**

From the  Department of Biological Sciences, Imperial College of Science, Technology, and Medicine, London SW7 2AY, United Kingdom and ^§ Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 23501-1980

Received for publication, May 24, 2002, and in revised form, June 12, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Human seminal plasma is a complex mixture of proteins, glycoproteins, peptides, glycopeptides, and prostaglandins secreted by organs of the male reproductive tract. The components of this fluid have been implicated in the suppression of immune response, agonistic effects on sperm-egg binding, and promotion of successful implantation of the human embryo. Fractionation followed by biophysical analyses revealed that free oligosaccharides constitute a major component of the total glycoconjugates within seminal plasma. Significant findings of our analyses include the following: (i) the concentration of free oligosaccharides is 0.3-0.4 mg/ml; (ii) mono- and difucosylated forms of the disaccharide lactose are major components; (iii) many of the remaining oligosaccharides are also rich in fucose and carry Lewis^x and/or Lewis^y epitopes; (iv) a subset of the oligosaccharides express the reducing end sequence (GlcNAc1-3/4Glc) not reported in human milk oligosaccharides; (v) oligosaccharides in seminal plasma exclusively express type 2 (Gal1-4GlcNAc) but not the type 1 sequences (Gal1-3GlcNAc) that predominate in human milk glycans; and (vi) the structural diversity of seminal plasma oligosaccharides is far less than human milk oligosaccharides. The agonistic effect of both fucose and fucosylated glycoconjugates on human sperm-egg binding in vitro suggests that fucosylated oligosaccharides may also promote fertilization in the female reproductive tract.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Human semen contains a variety of different cell types including sperm, neutrophils, monocytes, and lymphocytes (reviewed in Ref. 1). If incubated for a brief period of time, semen undergoes a process known as liquefaction that leads to clearing of this fluid and a partial loss of its viscosity (2). Centrifugation of partially liquefied semen leads to the separation of the cellular components from the viscous acellular fluid known as human seminal plasma (HSP).¹

HSP is more than simply a liquid medium for transporting sperm into the vagina. It is an extremely complex mixture of proteins, glycoproteins, peptides, glycopeptides, and prostaglandins secreted by the organs of the male reproductive tract (reviewed in Ref. 1). A plethora of different studies indicate that HSP supports sperm function, modulates maternal immune responses directed against sperm, and promotes successful implantation of the human embryo (reviewed in Refs. 1, 3, and 4). The components of HSP may therefore profoundly impact male fertility in the female reproductive tract.

Previous studies confirm that HSP contains a specific glycoprotein with immunomodulatory activities (5) now known as glycodelin-S (6). Glycodelin-S also promotes human sperm binding to homologous zona pellucida in the hemizona assay system (6). This intriguing combination of biological activities led us to investigate further the glycosylation of other components associated with HSP. By using fast atom bombardment (FAB) and electrospray (ES) mass spectrometry to screen HSP for novel glycoconjugates (7), we have made the surprising discovery that this fluid is also rich in free oligosaccharides. The only other human secretion known to contain a significant amount of free oligosaccharides is human milk (reviewed in Refs. 8 and 9). More than 90 distinct human milk oligosaccharides have been identified. Their structural heterogeneity is derived primarily from differential sialylation, fucosylation, branching, and polylactosamine chain extension (8, 9).

Several significant biological activities have been ascribed to human milk oligosaccharides. For example, many pathogens and bacterial toxins recognize terminal carbohydrate sequences associated with these glycans (reviewed in Ref. 10). Thus human milk oligosaccharides may block infection in infants by interfering with crucial adhesion and binding events essential for bacterial colonization and infection. A more recent study suggests that human milk oligosaccharides inhibit the binding of neutrophils activated with tumor necrosis factor to endothelial cells in vitro (9) and thus may modulate inflammatory events in vivo.

In this paper, we report the structural characterization of several families of free oligosaccharides present in HSP and show that they share some of the structural characteristics of human milk oligosaccharides, as well as having a number of unique features. The great majority are heavily fucosylated and contain structural motifs also present in the antennae of the N-linked oligosaccharides of glycodelin-S. The potential impact of these unusual free oligosaccharides on male reproductive function is discussed.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purification of Seminal Plasma Fractions-- Seminal plasma fractions were isolated from a range of healthy donors. Each sample was purified individually. After liquefaction, an equal volume of methanol was added to the semen, followed by sonication for 5 min and stirring at room temperature for 20 min. Methanol and chloroform were sequentially added to make the final composition (4:8:3) (v/v) in chloroform/methanol/water (11). The sonication procedure was repeated resulting in the majority of the proteins being precipitated. The sample was then centrifuged at 800 × g for 15 min. The supernatant was removed and saved. The precipitate was suspended in one-half the original extraction volume of chloroform/methanol/water (4:8:3), mixed, and centrifuged. The supernatant from each extraction was pooled and mixed with deionized water to make the final composition (4:8:5.6) in chloroform/methanol/water (11). The solution was then gently vortexed and allowed to stand for 30 min at room temperature, followed by centrifugation at 800 × g for 10 min. The upper layer was carefully removed and dried on a rotary evaporator.

The residue after drying was resuspended in 50% aqueous methanol and purified using a C₁₈ reversed phase Sep-Pak (Waters Associates) previously conditioned with methanol and 50% methanol washes. The void fraction was collected and lyophilized. The total hexose content of this fraction was compared with HSP using the phenol sulfuric acid assay (12). This fraction was dissolved in 1 ml of deionized water and stored at 20 °C in preparation for mass spectrometric studies. Further purification was carried out on a C₁₈ Sep-Pak cartridge conditioned with 5-ml washes of methanol, 5% acetic acid, and n-propyl alcohol followed by a final 10-ml wash of 5% acetic acid. The free oligosaccharides were eluted in 3 ml of 5% acetic acid.

Chemical Defucosylation-- Samples were incubated with 50 µl of 48% hydrogen fluoride (Aldrich) at 0 °C for 48 h. The reagent was removed by drying under a N₂ stream.

Deuteroreduction of Glycans-- Glycans were deuteroreduced using 200 µl of a 10 mg/ml solution of sodium borodeuteride in 2 M (aqueous) ammonium hydroxide. The reaction was allowed to proceed at room temperature for 2 h and was terminated by the dropwise addition of glacial acetic acid. The sample was dried under a N₂ stream and subjected to borate removal by repeated drying in the presence of 10% acetic acid in methanol.

Exoglycosidase Digestions-- The defucosylated glycans were incubated with the following enzymes: -L-fucosidase (almond meal EC 3.2.1.111, Glyko) 4 microunits in 100 µl of 50 mM sodium formate, pH 5.0; -galactosidase/lactase (Escherichia coli overproducer EC 3.2.1.23, Roche Molecular Biochemicals) 15 units in 100 µl of 50 mM ammonium formate and 50 mM potassium chloride pH 6.6; -galactosidase (bovine testis, EC 3.2.1.23, Roche Molecular Biochemicals) 10 milliunits in 100 µl of 50 mM ammonium formate pH 4.6; and -N-acetylhexosaminidase (bovine kidney, EC 3.2.1.30, Roche Molecular Biochemicals) 0.2 units in 100 µl of 50 mM ammonium formate buffer. The digestion with the -galactosidase from E. coli overproducer was performed for 48 h at 25 °C. All other enzyme digestions were carried out at 37 °C for 24 h, with fresh enzyme added after 12 h. Each reaction was terminated by boiling for 2 min.

Periodate Cleavage-- Glycans were deuteroreduced as described above. A solution of 35 mM sodium periodate in 100 mM ammonium acetate, pH 6.5, was prepared, and 100 µl were added to the glycans. This mixture was wrapped in foil and incubated at 0 °C overnight. The reaction was terminated by the addition of 2-3 µl of ethylene glycol and incubated at room temperature for 1 h. After drying under a N₂ stream, the products of periodate cleavage were reduced with 200 µl of a 10 mg/ml solution of sodium borohydride in 2 M ammonium hydroxide. This reaction was performed for 2 h, terminated by dropwise addition of glacial acetic acid, and dried under a N₂ stream. The sample was then subjected to borate removal, permethylation, and Sep-Pak clean-up using an acetonitrile gradient as described previously (7).

Methanolysis Experiments-- Permethylated glycans of D212 were defucosylated by mild acid hydrolysis (0.5 M methanolic HCl at room temperature). Aliquots of the methanolysis reaction were monitored every 2 min by FAB-MS analysis. The free hydroxyl groups were then remethylated using deuterated methyl iodide and subjected to linkage analysis.

Trimethylsilylation of Purified Glycans-- After Sep-Pak purification, methanolysis was performed on the sample. A solution of 1 M acetyl chloride in methanol was prepared by dissolving 100 µl of acetyl chloride in 1.3 ml of methanol. The glycans were dissolved in 200 µl of this solution and incubated overnight at 80 °C. The reaction was terminated by drying under a N₂ stream. The sample was re-N-acetylated by the sequential addition of 300 µl of methanol, 10 µl of pyridine, and 50 µl of acetic anhydride. After incubation for 15 min, the sample was dried under a N₂ stream. The sample was dissolved in 200 µl of Tri-sil-Z (Pierce) for 15 min before drying under a N₂ stream. The tetramethylsilane-derivatized glycans were dissolved in hexanes, vortexed, and centrifuged at 1000 rpm for 2 min. The supernatant was carefully transferred to a new tube, dried under a N₂ stream, and stored at 70 °C in preparation for GC-MS analysis.

Carbohydrate Standards for GC-MS Analysis-- Sophorose (Glc1-2Glc), laminaribose (Glc1-3Glc), lactose (Gal1-4Glc), and Man1-4Man were purchased from Sigma. Man1-2Man, Gal1-3Gal, Man1-3Man, and Gal1-4Gal were obtained from Dextra Laboratories, Ltd. The H-disaccharide (Fuc1-2Gal) was supplied by Accurate Chemicals. These disaccharides were used to determine the identity of the reducing hexose sugars present in seminal plasma sample D212. These standards were first deuteroreduced to label the reducing terminal sugars. This reaction was carried out prior to permethylation and subsequent conversion to partially methylated alditol acetates (13). GC-MS linkage analysis was performed on the carbohydrate standards. The retention times of the reducing hexoses in these standards were compared with those of the reducing terminal monosaccharides present in the sample. In separate runs, the seminal plasma sample was spiked with 500 pmol of each standard. Co-elution of reduced standards with the deuteroreduced hexoses in D212 led to identification of the reducing terminal sugars present in the seminal plasma-derived oligosaccharides.

Chemical Derivatization for FAB-MS, GC-MS, and CAD MS/MS Analysis-- Glycans were permethylated using the sodium hydroxide procedure and purified by Sep-Pak using a stepwise gradient of 0, 15, 35, 50, 75, and 100% aqueous acetonitrile as described previously (7). Partially methylated alditol acetates were prepared from the permethylated samples for GC-MS linkage analysis (13).

FAB-MS Analysis-- Fast atom bombardment-mass spectrometry was carried out using a ZAB 2SE 2FPD double-focusing mass spectrometer fitted with a cesium ion gun operated at 30 kV (7). Data were acquired and processed using VG Opus® software. Monothioglycerol was used as the matrix, and all permethylated samples were dissolved in methanol prior to loading (7).

GC-MS Analysis-- GC-MS analysis was carried out on a Fisons Instruments MD800 device. Separation was achieved using a RTX-5 fused silica capillary column (30 m × 0.25 mm internal diameter, Restek Corp.) The sample was dissolved in hexanes prior to on-column loading at 65 °C. The GC oven was held at 65 °C for 1 min before being increased at a rate of 8-290 °C/min.

Other MS Analyses-- Electrospray mass spectrometry (MS) and MS/MS were carried out using a Q-TOF (Micromass, UK) in positive ion mode. The permethylated glycans were dissolved in methanol at a concentration of ~10 pmol/µl. A sample flow rate of 10-30 nl/min was produced when a potential of 1.5 kV was applied to the nanoflow tip. The drying gas used was N₂ and the collision gas was argon, with the collision gas pressure maintained at 10⁴ millibar. Collision energies varied depending on the size of the carbohydrate, typically between 30 and 90eV.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Seminal Plasma Samples-- Seminal plasma samples obtained from two donors were examined in this study. Detailed structural data are reported for sample D212, and comparative data are provided for sample D112.

Structural Analysis Strategy-- The void volume fractions resulting from Sep-Pak purification of the seminal plasma samples were examined for their oligosaccharide content and composition. The void fraction obtained after the initial reverse phase separation contained 70% of the total hexose content present in HSP. Scheme 1 summarizes the structural analysis strategy employed. Briefly, methylated or deuteromethylated derivatives were characterized by FAB-MS, ES-MS/MS, and linkage analysis before and after deuteroreduction. These experiments were supplemented by similar examination of the products of exo-glycosidase digestions and hydrofluoric acid hydrolysates. The susceptibility of linear, deuteroreduced glycans to periodate oxidation was exploited to deduce linkage features of the oligosaccharides. All samples were purified on C₁₈ Sep-Pak cartridges prior to analysis, and derivatized oligosaccharides were recovered in the 35 and 50% aqueous acetonitrile fractions, as outlined under "Experimental Procedures."

View larger version (20K): [in this window] [in a new window]   Scheme 1.

FAB-MS Screening of D212 Identifies Several Families of Fucosylated Oligosaccharides-- Fig. 1 shows FAB data acquired from sample D212 oligosaccharides after deuteroreduction and permethylation. The upper and lower panels correspond to the 35 and 50% aqueous acetonitrile fractions, respectively. The low mass region of the 50% fraction (not shown) is similar to the 35% fraction except that the signals are of lower abundance. Assignments are given in Table I. Notable features of the data are as follows: (i) the major signals are attributed to fucosylated glycans, the three most abundant being m/z 709, 842, and 913 that correspond to monofucosylated HexHexNAc, difucosylated Hex₂, and monofucosylated Hex₂HexNAc, respectively; relatives of these glycans give the signals at m/z 494 (Hex₂), 535 (HexHexNAc), 668 (FucHex₂), 739 (Hex₂HexNAc), 883 (Fuc₂HexHexNAc), and 1087 (Fuc₂Hex₂HexNAc); (ii) two minor families of glycans consisting of mono-, di-, and tri-fucosylated Hex₃HexNAc and difucosylated Hex₂HexNAc₂ were also identified in the 50% aqueous acetonitrile fraction (signals m/z 1117, 1291, 1466, and 1333, respectively); (iii) the higher mass molecular ions (m/z 1536, 1710, 1885, 2160, 2334, 2509, 2610, 2784, 3233, 3408, 3583, and 3757) have compositions (see Table I) consistent with fucosylated glycans that have the Hex₂HexNAc unit at their reducing ends and are extended by up to four HexHexNAc repeats (i.e. polylactosamine type structure); remarkably, the longer glycans carry up to seven fucose residues; and (iv) minor A-type fragment ions were observed at m/z 812 (Fuc₂HexHexNAc⁺), m/z 638 (FucHexHexNAc⁺), and m/z 464 (HexHexNAc⁺) and were accompanied by signals indicating methanol loss. Corroborative data for these compositional assignments were obtained from deuteromethylated derivatives and from examination of permethylated non-reduced samples (data not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   FAB mass spectra of the free glycans of D212 isolated from human seminal fluid. Glycans were deuteroreduced, permethylated, and subjected to Sep-Pak clean-up. Upper panel, 35% acetonitrile fraction; lower panel, 50% acetonitrile fraction. The signals are assigned in Table I. All A-type ions assigned in Table I are very minor but nevertheless are reproducibly present in the various spectra acquired on this sample. The assignment of the minor signal at m/z 393 is corroborated by m/z 361 that corresponds to loss of methanol.

Linkage Analysis of the Free Glycans Isolated from Seminal Plasma Fraction D212 Reveals the Presence of Glucose as the Major Reducing Sugar-- Deuteroreduction prior to linkage analysis allowed the reducing terminal sugars to be detected in the linkage data (Table II). From these experiments it was clear that the main reducing monosaccharides associated with the HSP oligosaccharides are 4-linked glucose and its 3,4-linked counterpart (Fig. 2). Signals corresponding to 3-linked and 2-linked glucose as reducing terminal sugars were observed, although the latter sugar was found to be present only in minor amounts (Fig. 2). Other reducing end monosaccharides found in the seminal plasma glycans include 4-linked and 3,4-linked GlcNAc. Carbohydrate standards were used to distinguish between the different hexose residues.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   GC-MS analysis of the free glycans of D212 isolated from human seminal plasma. Deuteroreduction was carried out prior to linkage analysis. A, total ion chromatogram of D212 demonstrating the region where the deuteroreduced linked-glucose residues elute (see Table II), the peaks eluting at 17.20, 18.75, and 19.08 min are terminal Fuc, terminal Glc, and terminal Gal, respectively. The electron impact mass spectra of the peaks are eluting in the GC chromatogram at the positions of deuteroreduced 4-linked glucose (B) and deuteroreduced 3,4-linked glucose (C).

Linkage analysis data for sample D212 with no prior deuteroreduction are summarized in Table III. Key features of these data are as follows: (i) fucose, galactose, and GlcNAc are the major terminal sugars present, whereas GalNAc was only observed as a minor terminal component of the seminal plasma mixture and was not found in any linked form; (ii) a reduction in the signals corresponding to 2-linked Gal and 3,4-linked GlcNAc was observed after aqueous HF defucosylation and was coupled to an increase in terminal galactose and 4-linked GlcNAc; and (iii) minor amounts of differentially linked mannoses were observed that could be derived from N-glycans present in HSP. Additional information acquired from the analysis of trimethylsilyl ester methylglycosides of the total glycan population revealed that myoinositol was also present in high abundance (data not shown).

Characterization of Fucosylated Structures: Q-TOF MS/MS Analysis of the Permethylated Free Oligosaccharides Present in Deuteroreduced Oligosaccharides-- [M + Na]⁺ molecular ions in the mass range m/z 668-1710 were subjected to low energy, CAD MS/MS in order to define the sequences and, in some cases, the linkages of the glycans present in seminal fluid. The MS/MS spectra and fragmentation pathways for FucHex₂ (m/z 668), FucHexHexNAc (m/z 709), Fuc₂Hex₂ (m/z 842), Fuc₂HexHexNAc (m/z 883), FucHex₂HexNAc (m/z 913), Fuc₂Hex₂HexNAc (m/z 1087), Fuc₂Hex₃HexNAc₂ (m/z 1536), and Fuc₃Hex₃HexNAc₂ (m/z 1710) are shown in Figs. 3 and 4.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   CAD MS/MS spectra of the [M + Na]+ molecular ions of m/z 668 (A), m/z 709 (B), m/z 842 (C), and m/z 883 (D) of the permethylated glycans derived from the 35% acetonitrile fraction of deuteroreduced D212 isolated from human seminal plasma. The fragment ion m/z 489 in the MS/MS spectrum of molecular ion m/z 883 is a result of water loss from m/z 507. Other fragment ions are assigned in the inset.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   CAD MS/MS spectra of the [M + Na]+ molecular ions of m/z 913 (A), m/z 1087 (B), m/z 1536 (C), and m/z 1710 (D) of the permethylated free glycans derived from the 50% acetonitrile fraction of deuteroreduced D212 isolated from human seminal plasma. The fragment ion m/z 633 in A arises from -elimination of the fucose residue (m/z 707) followed by loss of the upper half of the GlcNAc residue via a ring cleavage mechanism (14). The fragment ions m/z 807 and 1430 in B and D, respectively, are similarly derived. The fragment ion m/z 415 in A results from the loss of a terminal galactose from m/z 633. The insets shown in C and D correspond to the major structure identified in each case. Signals attributable to the minor structure in C are explained in the text. Signals indicative of structural isomers of the inset shown in D are present at m/z 1073 (a fucose higher than m/z 899), m/z 867 (a fucose higher than m/z 693), and m/z 660 (a fucose lower than m/z 834). These signals indicate that a significant minority of the sample carries two fucoses attached to the reducing end trisaccharide.

Cleavage on either side of the glycosidic oxygen provided information that was useful for sequence assignment, and these signals were prevalent in all MS/MS spectra. Cross-ring fragments determining linkage position were generally weaker, although they were observed in the MS/MS spectra of lower mass components; for example, the MS/MS spectrum of m/z 913 contains a signal m/z 329, confirming the 4-linkage between terminal Gal and sub-terminal GlcNAc.

Previous mass spectrometric analyses have shown that when a substituent is 3-linked to a HexNAc unit, it is readily eliminated (14). A large signal corresponding to elimination of fucose from GlcNAc (m/z 503) was observed following CAD MS/MS analysis of the mono-fucosylated trisaccharide FucHexHexNAc (Fig. 3B), indicating that the fucose residue was originally 3-linked in this case. CAD MS/MS analysis also on mono-fucosylated Hex₂HexNAc (m/z 913, Fig. 4A) revealed that the fucose residue is 1-3 linked to the GlcNAc, whereas the second fucose is preferentially attached to the terminal galactose residue in the difucosylated form (m/z 1087, Fig. 4B).

The spectrum of m/z 1536 (Fuc₂Hex₃HexNAc₂) (Fig. 4C) indicates that that the majority of this component is fucosylated at each of the GlcNAc residues as shown in the inset. Thus, major signals are observed at m/z 660 and 899 that correspond to mono-fucosylated non-reducing end and mono-fucosylated reducing end structures, respectively, resulting from cleavage at the GlcNAc-Gal glycosidic linkage in the center of the backbone (see Fig. 4C). The very minor signals at m/z 834 and 725 are the corresponding fragment ions for an isomeric structure in which the two fucose residues are attached to the non-reducing disaccharide to give a Lewis^y epitope. A signal corresponding to -elimination of fucose from the major fragment ion m/z 899 (m/z 693), together with the presence of an ion corresponding to non-reducing Hex (m/z 259), provide confirmatory evidence for the majority of fucose being linked to the two GlcNAc residues in Lewis^x arrangements. Interpreting the MS/MS spectrum of m/z 1710 (Fuc₃Hex₃HexNAc₂) (Fig. 4D) in a similar manner allows us to arrive at the conclusion that the majority of the glycans with this composition carry fucose on the non-reducing galactose and the adjacent GlcNAc residue, with the third fucose being attached to the other GlcNAc residue (see inset in Fig. 4D). Conversely, a portion of the glycans have a fucose residue attached at each GlcNAc, with the third fucose being located on either the 3-linked galactose or the reducing terminal glucose (see legend to Fig. 4D).

Comparison of MS/MS data with that derived from authentic material indicated that the molecular ions m/z 668, 709, 842, and 883 likely correspond to 3'-fucosyllactose, Lewis^X, difucosyllactose, and Lewis^y, respectively (data not shown).

Confirmation of Inter-saccharide Linkages Using Methanolysis and Periodate Cleavage-- Corroborative evidence for the positions of attachment of the fucose residues was provided by mild methanolysis experiments. Permethylated glycans of D212 were defucosylated using mild acid hydrolysis (0.5 M methanolic HCl at room temperature) and monitored by FAB-MS. The free hydroxyl groups were re-methylated using deuterated methyl iodide, and linkage analysis was carried out to deduce where the fucoses had been attached. This structural feature can be identified by a characteristic mass shift of the fragment ion in the electron impact mass spectrum. Additional signals at 121 and 165 Da were observed in the electron impact mass spectrum of terminal Gal (data not shown), verifying that fucose had been attached previously at the 2-position of galactose. Comparison of linkage data before and after methanolysis indicates that the loss of fucose from 3,4-linked glucose is accompanied by an increase in 4-linked glucose. The EI-MS data for 4-linked Glc and 4-linked GlcNAc unambiguously confirm that fucose is 3-linked to these sugars in both cases based upon the presence of a new signal at m/z 236. No new signals were observed from the EI-MS of 3-linked Glc and 2-linked Glc, indicating that these components were not originally fucosylated.

Mild periodate oxidation will cleave the vicinal hydroxyl groups present in linear sugar structures. Therefore the reducing sugars of deuteroreduced glycans are susceptible to this reagent. Purified glycans were subjected to periodate oxidation (35 mM concentration) followed by reduction, permethylation, and Sep-Pak clean-up in preparation for FAB-MS analysis. Fast atom bombardment of the periodate products resulted in new signals representing loss of the relevant substituents, therefore confirming linkage positions. Significant peaks at m/z 780 and 954 (from m/z 913 and 1087, respectively) indicated that the deuteroreduced glucose in this glycan family was not 2-linked and was instead 3- or 4-linked.

Exoglycosidase and Chemical Digestions-- To establish the anomeric configurations of the glycan residues present in D212, a series of exoglycosidases were employed. The -linkage of the fucose residues was determined after digestion of the seminal plasma glycans with -L-fucosidase prior to permethylation and FAB-MS analyses.

HF-treated glycans were further digested with lactase resulting in a marked decrease in m/z 477 (Hex₂), substantiating the presence of lactose. Subsequent -galactosidase and -N-acetylhexosaminidase digests confirmed the -linkages of galactose and GlcNAc residues.

Comparison of D212 with Seminal Plasma Samples Obtained from Different Donors-- FAB-MS strategies were employed on sample D112, revealing a similar glycan profile to that of D212 but with inherently lower levels of fucosylation. The absence of molecular ions m/z 842, 883, 1087, 1710, and 1885 in the FAB-MS data and of signals in the linkage data corresponding to 2-linked galactose would imply that the gene for -1,2-fucosyltransferase is not expressed in the reproductive glands of this individual.

Structural Conclusions-- Taking into consideration the FAB-MS, linkage, CAD MS/MS, periodate, methanolysis, and exoglycosidase data, we conclude that human seminal plasma contains several families of highly fucosylated free glycans, with the sequences shown in Fig. 5. The main features are as follows. (i) These glycans are composed of differing backbones with the most abundant consisting of either lactose-based or novel Gal1-4GlcNAc1-3/4Glc sequences decorated with varying levels of fucosylation. (ii) The fucoses are attached to the Gal and GlcNAc residues giving both Lewis^x and Lewis^y terminated structures. (iii) The largest glycan observed is an octadecamer with the composition Fuc₇Hex₆HexNAc₅ (m/z 3757, Fig. 1B and Table I) containing the Gal1-4GlcNAc1-3/4Glc sequence at the reducing terminal extended by four N-acetyllactosamine repeats, with an overall level of fucosylation consistent with substitution of each GlcNAc residue and both terminal sugars.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Proposed structures of the major free oligosaccharides present in deuteroreduced D212 isolated from human seminal plasma. The ambiguity with respect to the linkage of the reducing end glucose in structures that are not fucosylated at this sugar arises from the observation of both 3- and 4-linked glucoses in the linkage analysis. Structures IXa and Xa are the major constituents giving m/z 1536 and 1710, respectively. Structure Xb is the most likely Lewisx-containing isomer of structure Xa, although the observation of a trace amount of 2,3-Gal in the linkage analysis suggests that a tiny portion of the sample probably carries the third fucose on the central galactose rather than the reducing-end glucose. Similarly, we have not ruled out the possibility of the presence of the analogous isomer of structure XI (m/z 1885) in which the central galactose is fucosylated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To our knowledge this study is the first revealing the existence of free oligosaccharides in HSP. Thus both human milk and HSP are secretions that contain free oligosaccharides. However, there are several significant differences between milk and HSP oligosaccharides revealed by the current structural analyses: (i) the concentration of free oligosaccharides is greater in human milk than HSP (5-8 versus 0.3-0.4 mg/ml) (9); (ii) sialylated oligosaccharides are very minor components in HSP (data not shown) but represent 25-30% of the total human milk glycans (15); (iii) the majority of complex HSP oligosaccharides are fucosylated; (iv) a subset of HSP oligosaccharides express the reducing end sequence (GlcNAc1-3/4Glc) not reported in human milk oligosaccharides; (v) HSP oligosaccharides exclusively express type 2 (Gal1-4GlcNAc) but not the type 1 sequences (Gal1-3GlcNAc) that predominate in human milk oligosaccharides (9); and (vi) the structural diversity of human milk oligosaccharides greatly exceeds that of HSP oligosaccharides (16).

The pathway for the synthesis of HSP and human milk oligosaccharides may share some common features, however. -Galactosyltransferase and -fucosyltransferase activities associated with free oligosaccharide synthesis are present in human milk (17, 18). Similar enzymatic activities are also present in HSP (19, 20). The properties of the HSP-associated galactosyltransferase include the following: (i) sensitivity to -lactalbumin (20), a modifier protein in mammalian milk (21) that shifts the substrate acceptor specificity of breast milk galactosyltransferase from GlcNAc to Glc (22); (ii) origin primarily in the prostate and epididymis (19, 20); and (iii) androgen dependence (20). A possible functional linkage is the observation that human sperm penetration through cervical mucus is positively correlated with this HSP-associated -galactosyltransferase activity (23).

Another interesting finding is that the level of HSP-associated -fucosyltransferase activity greatly exceeds the -galactosyltransferase activity in vitro (24). This observation is consistent with the content of fucosylated oligosaccharides present in HSP. This -fucosyltransferase activity originates primarily in the prostate and is also androgen-dependent (25).

There is currently no evidence suggesting that other mammalian species express free oligosaccharides in their seminal plasma. However, there are data supporting the existence of glycosyltransferases and associated biosynthetic proteins in rat seminal plasma. Both a substantial -galactosyltransferase activity and an -lactalbumin homologue are present in rat seminal plasma (26). Rat seminal plasma also contains very substantial amounts of -fucosyltransferase activity (27). These enzymes are postulated to function in the modification of sperm-surface glycoproteins (27), but based on the current evidence such enzymes could be involved in free oligosaccharide synthesis.

The function of HSP oligosaccharides in the male and/or the female reproductive systems is unknown. Because humans do not inject semen directly into the uterus, sperm and other seminal plasma components must traverse the cervical mucin plug to enter this organ (reviewed in Ref. 28). It may be easier for smaller components like free oligosaccharides to move through this plug at midcycle to influence events within the uterus and the oviduct. Support of sperm function in the uterus and oviduct would certainly be physiologically relevant. Of the several million sperm present in human semen, at most a few hundred arrive in the ampulla of the oviduct where fertilization takes place (reviewed in Ref. 1). Indeed, a very great mystery is how natural fertilization occurs in the presence of such low concentrations of sperm compared with those required for successful in vitro fertilization or sperm binding assays. The logical reason is that male and female factors operating within the vagina and oviduct facilitate this process.

Possible supportive effects of free oligosaccharides on sperm function include the following: (i) increasing sperm longevity by delaying hyperactivation; (ii) modifying sperm motion parameters that increase fertility, especially progressive motility; and (iii) promoting sperm binding to eggs.

Human sperm display decreased hyperactivation and increased progressive motility following exposure to human cervical mucins in vitro (29). The major O-glycans associated with human midcycle cervical mucins are terminated with Lewis^x and Lewis^y sequences (30), as are HSP oligosaccharides. Human sperm binding to the zona pellucida is increased by 20% in the hemizona assay in the presence of fucose (1 mg/ml) but not other monosaccharides (31). Similarly, glycodelin-S, a HSP glycoprotein also terminated with Lewis^x and Lewis^y sequences, increases sperm binding in the hemizona assay by 50% at physiological concentrations (6). The expression of fucosylated sequences on free oligosaccharides, mucins, and glycoproteins may promote sperm-egg binding in the human oviduct.

The oligosaccharides associated with HSP could also play a pivotal role in blocking immune/inflammatory cell reactions in the male and female reproductive systems. Except for sperm, leukocytes are the most prevalent cell type present in HSP from fertile males. These leukocytes are primarily neutrophils, with lower numbers of monocytes and T cells (reviewed in Ref. 32). Leukocytospermia is a condition characterized by excessive numbers of leukocytes in semen (reviewed in Ref. 33). Elevation in leukocytes above a certain threshold is associated with male infertility (32). There is also a very profound influx of leukocytes into the vagina and cervix following intercourse, an event referred to as the leukocyte reaction (34). The majority of the invasive cells are neutrophils, with minor amounts of natural killer cells and monocytes (34). Human sperm express carbohydrate sequences that are recognized by natural killer cells (35), so they are likely protected from this type of lymphocyte. However, the factors protecting sperm from other immune and inflammatory cell types in semen and the uterus are not very well defined.

There is some good evidence that prostate vesicles (prostasomes) present in HSP scavenge reactive oxygen species produced by neutrophils and monocytes (36). Therefore, prostasomes may protect sperm from the toxic by-products of neutrophil metabolism. Other investigators suggest that prostaglandins (PGE₂ and 19-hydroxy-PGE) present in HSP play a crucial role in the suppression of leukocytes (3). However, incubation of the neutrophil model cell line U937 with a seminal plasma fraction enriched in PGE₂ and 19-hydroxy-PGE had no effect on the release of the immunosuppressive cytokine interleukin-10 at the highest concentration tested (0.1% of the seminal plasma concentration) (37). By contrast, incubation of U937 cells with media containing HSP diluted to the same extent induces substantial interleukin-10 release from U937 cells (37). This result implies that other components within HSP are likely responsible for the major suppressive effect of this secretion. One factor implicated in this immunomodulation is glycodelin-S, a glycoprotein that shares terminal Lewis^x and Lewis^y sequences with the HSP oligosaccharides (6).

Human milk oligosaccharides block the binding of many pathogens or their toxins to colonic epithelial cells in vitro (reviewed in Refs. 9 and 10). Several of these crucial interactions are inhibited by fucosylated oligosaccharides present in this secretion. HSP oligosaccharides could also block infection with pathogens responsible for urogenital tract infections. Fucosylated glycans have been implicated in infection with human T-cell lymphotrophic virus, type I, and human immunodeficiency virus (38, 39). Additional study will be required to determine whether HSP oligosaccharides confer any protective effect against pathogens in the male and/or female reproductive systems.

In summary, free oligosaccharides of highly restricted sequence heterogeneity are expressed in the HSP of fertile men. The presence of a predominant modification of the complex glycans (fucosylation) suggests potential functional significance especially when linked to previous data collected in the human model. Further investigation will be required to define the precise physiological roles of these free oligosaccharides in human reproduction.

	FOOTNOTES

^* This work was supported by the Biotechnology and Biological Sciences Research Council and the Wellcome Trust (to A. D. and H. R. M.), National Institutes of Health Grant HD35652 (to G. F.  C.), and Jeffress Research Grant J-584 (to M. S. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence may be addressed. Tel.: 44-207-225-5219; Fax: 44-207-225-0458; E-mail: h.morris@ic.ac.uk.

To whom correspondence may be addressed. Tel.: 44-207-225-5219; Fax: 44-207-225-0458; E-mail: a.dell@ic.ac.uk.

^** To whom correspondence may be addressed. Tel.: 757-446-5653; Fax: 757-624-2270; E-mail: clarkgf@evms.edu.

Published, JBC Papers in Press, June 12, 2002, DOI 10.1074/jbc.M205152200

	ABBREVIATIONS

The abbreviations used are: HSP, human seminal plasma; FAB-MS, fast atom bombardment mass spectrometry; ES, electrospray; GC, gas chromatography; MS, mass spectrometry; Q-TOF, quadrupole orthogonal acceleration time of flight mass spectrometer; CAD, collisionally activated decomposition; PG, prostaglandin.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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