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Glycomics and Proteomics Analyses of Mouse Uterine Luminal Fluid Revealed a Predominance of Lewis Y and X Epitopes on Specific Protein Carriers

  • Chu-Wei Kuo
    Affiliations
    Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 11490, Taiwan

    Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
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  • Chin-Mei Chen
    Affiliations
    Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
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  • Ying-Chu Lee
    Affiliations
    Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
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  • Sin-Tak Chu
    Correspondence
    To whom correspondence may be addressed: Inst. of Biological Chemistry, Academia Sinica, 128 Academia Rd. Sec 2, Nankang, Taipei 11529, Taiwan. Fax: 886-2-27889759
    Affiliations
    Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
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  • Kay-Hooi Khoo
    Correspondence
    To whom correspondence may be addressed: Inst. of Biological Chemistry, Academia Sinica, 128 Academia Rd. Sec 2, Nankang, Taipei 11529, Taiwan. Fax: 886-2-27889759
    Affiliations
    Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan

    National Research Program for Genomic Medicine Core Facilities for Proteomics and Glycomics at Academia Sinica, Taipei 11529, Taiwan
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Open AccessPublished:October 21, 2008DOI:https://doi.org/10.1074/mcp.M800320-MCP200
      Sperm motility and maturation are known to be affected by a host of factors encountered en route in both male and female genital tracts prior to fertilization. Using a concerted proteomics and glycomics approach with advanced mass spectrometry-based glycan sequencing capability, we show in this work that 24p3, an abundant mouse uterine luminal fluid (ULF) glycoprotein also called lipocalin 2 (Lcn2), is highly fucosylated in the context of carrying multiple Lewis X and Y epitopes on complex type N-glycans at its single glycosylation site. The predominance of Lewis X/Y along with Neu5Acα2–6 sialylation was found to be a salient feature of the ULF glycome, and several other protein carriers were additionally identified including the highly abundant lactotransferrin, which is N-glycosylated at two sites, both with a similar range of highly fucosylated N-glycans. A comparative glycomics analysis of the male genital tract fluids revealed that there is a gradient of glycomic complexity from the cauda to caput regions of the epididymis, varying from high mannose to sialylated complex type N-glycans but mostly devoid of fucosylation. The seminal vesicle fluid glycome, on the other hand, carries equally abundant multimeric Lewis X structures but is distinctively lacking in additional fucosylation of the terminal galactose to give the Lewis Y epitope typifying the glycome of female ULF. One-dimensional shotgun proteomics analysis identified over 40 proteins in the latter, many of which are reported for the first time, and a majority are notably involved in immune defense and antigen processing. Further sperm binding and motility assays suggest that the Lewis X/Y epitopes do contribute to the sperm motility-enhancing activity of 24p3, whereas lactotransferrin is largely inactive in this context despite being similarly glycosylated. These findings underline the importance of glycoproteomics in delineating both the specific glycan structures and their carriers in assigning glycobiological functions.
      The advent of functional genomics and proteomics has facilitated systematic investigations into the key events from spermatogenesis to fertilization in reproductive glycobiology using the mouse model (
      • Wassarman P.M.
      • Jovine L.
      • Litscher E.S.
      A profile of fertilization in mammals.
      ). Although the general importance of glycosylation is well recognized (
      • Dell A.
      • Morris H.R.
      • Easton R.L.
      • Patankar M.
      • Clark G.F.
      The glycobiology of gametes and fertilization.
      ,
      • Tulsiani D.R.
      • Yoshida-Komiya H.
      • Araki Y.
      Mammalian fertilization: a carbohydrate-mediated event.
      ), specific structural details of the implicated glycotopes are surprisingly lacking. The best studied glycotopes are those on the zona pellucida glycoproteins (
      • Tulsiani D.R.
      Structural analysis of the asparagine-linked glycan units of the ZP2 and ZP3 glycoproteins from mouse zona pellucida.
      ,
      • Easton R.L.
      • Patankar M.S.
      • Lattanzio F.A.
      • Leaven T.H.
      • Morris H.R.
      • Clark G.F.
      • Dell A.
      Structural analysis of murine zona pellucida glycans. Evidence for the expression of core 2-type O-glycans and the Sd(a) antigen.
      ,
      • Wassarman P.M.
      Zona pellucida glycoproteins.
      ) thought to mediate sperm binding to oocytes. Virtually nothing is known about the potential roles mediated by glycosylation as the maturing spermatozoa moves from storage in epididymis through ejaculation during copulation into the uterine tract, encountering en route a myriad of secretory glycoproteins within the bathing fluids. Adsorption or interactions with various glycotopes presented by these secretory glycoproteins may have profound effect in modulating the characteristics of sperm glycocalyx (
      • Schroter S.
      • Osterhoff C.
      • McArdle W.
      • Ivell R.
      The glycocalyx of the sperm surface.
      ,
      • Tulsiani D.R.
      • Orgebin-Crist M.C.
      • Skudlarek M.D.
      Role of luminal fluid glycosyltransferases and glycosidases in the modification of rat sperm plasma membrane glycoproteins during epididymal maturation.
      ) and, through that, its fertilizing potential. The observation that glycotopes are often involved in eliciting antisperm immune responses (
      • Kurpisz M.
      • Alexander N.J.
      Carbohydrate moieties on sperm surface: physiological relevance.
      ) and the many reported incidences of their dynamic modulation in association with sperm fertility underscore the importance of precise structural and functional knowledge in efforts directed toward developing them as targets for immunocontraceptive vaccines (
      • McCauley T.C.
      • Kurth B.E.
      • Norton E.J.
      • Klotz K.L.
      • Westbrook V.A.
      • Rao A.J.
      • Herr J.C.
      • Diekman A.B.
      Analysis of a human sperm CD52 glycoform in primates: identification of an animal model for immunocontraceptive vaccine development.
      ).
      In human, glycodelin is a major uterine fluid and seminal plasma glycoprotein with gender-specific glycosylation and contraceptive properties (
      • Seppala M.
      • Taylor R.N.
      • Koistinen H.
      • Koistinen R.
      • Milgrom E.
      Glycodelin: a major lipocalin protein of the reproductive axis with diverse actions in cell recognition and differentiation.
      ,
      • Yeung W.S.
      • Lee K.F.
      • Koistinen R.
      • Koistinen H.
      • Seppala M.
      • Ho P.C.
      • Chiu P.C.
      Glycodelin: a molecule with multi-functions on spermatozoa.
      ). It belongs to the lipocalin superfamily characterized by shared three-dimensional structure and the ability to bind small hydrophobic molecules (
      • Akerstrom B.
      • Flower D.R.
      • Salier J.P.
      Lipocalins: unity in diversity.
      ). The progesterone-regulated glycodelin-A of uterine origin potently and dose-dependently inhibits human sperm-egg binding through interaction with sperm, whereas differently glycosylated glycodelin-S from seminal plasma has no such effect (
      • Morris H.R.
      • Dell A.
      • Easton R.L.
      • Panico M.
      • Koistinen H.
      • Koistinen R.
      • Oehninger S.
      • Patankar M.S.
      • Seppala M.
      • Clark G.F.
      Gender-specific glycosylation of human glycodelin affects its contraceptive activity.
      ). The latter is unusually fucose-rich, carrying a high abundance of Lewis X (Lex)
      The abbreviations used are: Lex, Lewis X; Ley, Lewis Y; AAL, A. aurantia lectin; DES, diethylstilbestrol; EF, epididymal fluid; LacNAc, N-acetyllactosamine; SVF, seminal vesicle fluid; ULF, uterine luminal fluid; Lcn2, lipocalin 2; Fuc, fucose; Hex, hexose; HexNAc, N-acetylhexosamine; HM, HEPES medium; NCBInr, National Center for Biotechnology Information non-redundant; PID, precursor ion discovery; GC, gas chromatography; Lea, Lewis A; Leb, Lewis B; 1D, one-dimensional; NeuAc (Neu5Ac), N-acetylneuraminic acid; Neu5Ac, 5-N-acetylneuraminic acid; Neu5Gc, 5-N-glycolylneuraminic acid; LPS, lipopolysaccharide.
      1The abbreviations used are: Lex, Lewis X; Ley, Lewis Y; AAL, A. aurantia lectin; DES, diethylstilbestrol; EF, epididymal fluid; LacNAc, N-acetyllactosamine; SVF, seminal vesicle fluid; ULF, uterine luminal fluid; Lcn2, lipocalin 2; Fuc, fucose; Hex, hexose; HexNAc, N-acetylhexosamine; HM, HEPES medium; NCBInr, National Center for Biotechnology Information non-redundant; PID, precursor ion discovery; GC, gas chromatography; Lea, Lewis A; Leb, Lewis B; 1D, one-dimensional; NeuAc (Neu5Ac), N-acetylneuraminic acid; Neu5Ac, 5-N-acetylneuraminic acid; Neu5Gc, 5-N-glycolylneuraminic acid; LPS, lipopolysaccharide.
      and Lewis Y (Ley) epitopes (±Fucα1–2Galβ1–4(Fucα1–3)GlcNAc) on core fucosylated biantennary complex type N-glycan structures instead of the fucosylated or sialylated N,N′-diacetyllactosediamine antenna found on glycodelin-A. The expression of glycodelin mRNA in the female and male rodent genital tracts has been demonstrated (
      • Keil C.
      • Husen B.
      • Giebel J.
      • Rune G.
      • Walther R.
      Glycodelin mRNA is expressed in the genital tract of male and female rats (Rattus norvegicus).
      ), but nothing is known of the functional gene product or its glycosylation. A distinct 24p3 glycoprotein belonging likewise to the lipocalin superfamily, also called lipocalin 2 (Lcn2), has been purified from mouse uterine luminal fluid (
      • Chu S.T.
      • Huang H.L.
      • Chen J.M.
      • Chen Y.H.
      Demonstration of a glycoprotein derived from the 24p3 gene in mouse uterine luminal fluid.
      ) and additionally shown to be expressed in the epididymis (
      • Chu S.T.
      • Lee Y.C.
      • Nein K.M.
      • Chen Y.H.
      Expression, immunolocalization and sperm-association of a protein derived from 24p3 gene in mouse epididymis.
      ). Monosaccharide composition analysis of 24p3 from the uterine fluid has revealed a very high fucose content along with other glycosyl residues indicative of N-glycosylation. No specific structures have been defined, nor is it clear whether the epididymal form is differently glycosylated.
      Fucosylation is widely implicated in mouse reproductive physiology from epididymal sperm maturation to blastocyst implantation (
      • Kimber S.J.
      • Spanswick C.
      Blastocyst implantation: the adhesion cascade.
      ,
      • Ram P.A.
      • Cardullo R.A.
      • Millette C.F.
      Expression and topographical localization of cell surface fucosyltransferase activity during epididymal sperm maturation in the mouse.
      ). In particular, expression of α2-fucosylated glycans on the uterine epithelium is known to be dynamically regulated in concert with the estrous cycle during which the α2-fucosyltransferase mRNA level is high after estrogenic stimulation and ovulation but becomes negligible after implantation (
      • Kimber S.J.
      • Stones R.E.
      • Sidhu S.S.
      Glycosylation changes during differentiation of the murine uterine epithelium.
      ). Such hormonal regulation of glycosylation is expected to extend to the synthesized and secreted components. A gradient of fucosyltransferase activity has also been demonstrated to exist from the caput to cauda epididymis that parallels the apparent acquisition by the maturing spermatozoa of surface α2-fucosylated glycans and the ability to fertilize during epididymal passage (
      • Ram P.A.
      • Cardullo R.A.
      • Millette C.F.
      Expression and topographical localization of cell surface fucosyltransferase activity during epididymal sperm maturation in the mouse.
      ,
      • Tulsiani D.R.
      • Skudlarek M.D.
      • Holland M.K.
      • Orgebin-Crist M.C.
      Glycosylation of rat sperm plasma membrane during epididymal maturation.
      ). Optimal exposure and density of these glycotopes are dynamically remodeled upon further interactions with uterine secretory components as the sperm attains timely capacitation in the female reproductive tract. Nonetheless the respective functions of specific fucosylated epitopes remain unsolved. Deficiency in the expression of α2-fucosylated glycans on either the epididymal or uterine epithelia alone in mice with targeted deletions of the FUT1 or FUT2 α2-fucosyltransferase, respectively, did not seem to impair fertility (
      • Domino S.E.
      • Zhang L.
      • Gillespie P.J.
      • Saunders T.L.
      • Lowe J.B.
      Deficiency of reproductive tract α(1,2)fucosylated glycans and normal fertility in mice with targeted deletions of the FUT1 or FUT2 α(1,2)fucosyltransferase locus.
      ). On the other hand, mice with a null mutation in the FX locus, which encodes an enzyme involved in the de novo pathway for GDP-fucose synthesis, were infertile when reared in the absence of fucose (
      • Smith P.L.
      • Myers J.T.
      • Rogers C.E.
      • Zhou L.
      • Petryniak B.
      • Becker D.J.
      • Homeister J.W.
      • Lowe J.B.
      Conditional control of selectin ligand expression and global fucosylation events in mice with a targeted mutation at the FX locus.
      ), thus supporting an essential role for fucosylation in reproduction. Likewise about half of the Golgi GDP-fucose transporter-deficient mice were found to be infertile (
      • Hellbusch C.C.
      • Sperandio M.
      • Frommhold D.
      • Yakubenia S.
      • Wild M.K.
      • Popovici D.
      • Vestweber D.
      • Grone H.J.
      • von Figura K.
      • Lubke T.
      • Korner C.
      Golgi GDP-fucose transporter-deficient mice mimic congenital disorder of glycosylation IIc/leukocyte adhesion deficiency II.
      ).
      It is argued herein that the role of fucosylation in particular and glycosylation in general cannot be rationally delineated without a prior knowledge of the precise glycostructures involved and presented ensemble by the constituent fluid proteomes. By adopting a concerted proteomics and glycomics analyses, we report here the identification of several secretory components not known previously to be associated with the uterine luminal fluid and show that Lex and Ley are the major fucosylated epitopes of its constitutive glycoproteins including 24p3 and lactotransferrin. The same Ley epitope, which is also the predominant glycotope on human glycodelin-S but not glycodelin-A, is further demonstrated here to be involved in sperm/24p3 interactions. Interestingly only Lex but not Ley was found on the male seminal vesicle fluid glycoproteins, whereas similar glycomics profiling of the epididymal fluids revealed that neither of the fucosylated glycotopes is presented to the maturing sperm in these sections of the male reproductive tracts.

      DISCUSSION

      Despite recent technical advances in both MS instruments and front end sample preparation, glycoproteomics analysis remains daunting. In principle, all requisite information for unambiguous identification of the protein carrier, its glycosylation site(s), and the exact glycan structures encompassing a full range of heterogeneous glycoforms can be obtained from detailed MS/MS analysis of the glycopeptides. In practice, however, this premise is limited by detecting the glycopeptides in the first place and the subsequent difficulties in obtaining a full range of sequence-informative MS/MS fragment ions pertaining to both the peptide and glycan sequence at sufficiently high sensitivity. In particular, glycan sequencing that would unambiguously determine its linkage-specific substituents and branching pattern is usually not feasible by MS/MS analysis at the glycopeptide level. Instead it is more readily accomplished by analyzing the permethylated derivatives of the released glycans either by ion trap-based MSn (
      • Ashline D.J.
      • Lapadula A.J.
      • Liu Y.H.
      • Lin M.
      • Grace M.
      • Pramanik B.
      • Reinhold V.N.
      Carbohydrate structural isomers analyzed by sequential mass spectrometry.
      ) or, as in our case, by complementary low and high energy MALDI CID MS/MS (
      • Yu S.Y.
      • Wu S.W.
      • Khoo K.H.
      Distinctive characteristics of MALDI-Q/TOF and TOF/TOF tandem mass spectrometry for sequencing of permethylated complex type N-glycans.
      ,
      • Fan Y.Y.
      • Yu S.Y.
      • Ito H.
      • Akihiko K.
      • Sato T.
      • Lin C.H.
      • Yu L.C.
      • Narimatsu H.
      • Khoo K.H.
      Identification of further elongation and branching of dimeric type 1 chain on lactosylceramides from colonic adenocarcinoma by tandem mass spectrometry sequencing analyses.
      ,
      • Wu A.M.
      • Khoo K.H.
      • Yu S.Y.
      • Yang Z.
      • Kannagi R.
      • Watkins W.M.
      Glycomic mapping of pseudomucinous human ovarian cyst glycoproteins: identification of Lewis and sialyl Lewis glycotopes.
      ). With additional exoglycosidase digestion and GC-MS analysis, we have unambiguously defined the ULF glycome as comprising core fucosylated complex type N-glycans with multiple Lex and Ley epitopes to account for its high Fuc content along with a variable amount of Neu5Acα2–6-sialylated antenna. This overall glycomic pattern is reflective of its constituent glycoproteins including the two most abundant ones, 24p3 and lactotransferrin.
      Because 24p3 was purified to homogeneity and N-glycans were released thereof, attribution of the glycosylation profile was not an issue. We have additionally found that only one of two potential sites was N-glycosylated while confirming that its N terminus was indeed modified by pyroglutamation. In the case of lactotransferrin, direct LC-MS/MS analysis of the glycopeptides in conjunction with identification of the de-N-glycosylated peptides by MALDI MS/MS have collectively verified the two occupied N-glycosylation sites, both carrying the implicated N-glycan structures. However, identification of other glycopeptides was less conclusive. We have to resort instead to a commonly used approach, namely to first isolate the highly fucosylated glycopeptide pool by lectin affinity and subsequently identify the proteins based on MS/MS analysis of the de-N-glycosylated peptides with a requisite Asn to Asp conversion. Not all of the 10 ULF proteins thus identified were in the list of the identified ULF proteins by shotgun analysis of the total tryptic digests. This would be expected because the lectin affinity capture would give an enrichment effect for some of the less abundant proteins carrying highly fucosylated N-glycan structures. Glycomics analysis of this lectin-captured pool of glycopeptides indicated that all N-glycan structures released thereof contained at least three Fuc residues, thus containing a minimum of one terminal Lewis epitope in addition to core fucosylation. This largely rids the identified de-N-glycosylated peptides of false positives due to nonspecific bindings commonly associated with affinity capture.
      Conversely not all abundant ULF proteins identified by shotgun proteomics analysis appeared to carry the multifucosylated N-glycan structures, although false negatives could be contributed by many factors including random misses. By high stringency statistical filtering, we reported in this work 44 ULF proteins deemed identified with high confidence. A problem with analysis of the ULF proteome, as in the case of plasma, was the predominant abundance of a few major components such as complement C3 and lactotransferrin, which precluded more proteins to be identified by a single dimensional shotgun analysis of non-fractionated samples despite the use of a high performance instrument. An initial attempt at two-dimensional gel separation followed by spotwise identification also did not result in a more comprehensive coverage of the ULF proteome. Likewise the analysis of the glycopeptides was dominated by repeated MS/MS on the glycoforms of lactotransferrin. Because the main focus of this work was centered on detailed glycomics analysis and to obtain a simplistic overview of the prevalent characteristics of the ULF glycoproteome, we have not delved further into the depth of proteomic coverage.
      For the more abundant proteins, the three major groupings according to the apparent biological processes in which they may be involved are: 1) immune response, including proteins involved in complement pathways such as complement C3, complement factor H, complement factor B, and modulators involved in the innate immune response such as CD14 antigen, lipopolysaccharide (LPS)-binding protein, and peptidoglycan recognition protein; 2) metabolism, including several enzymes such as aminopeptidase N, α 3A chain of laminin-5, alkaline phosphatase, chitinase 3-like protein 1, and cathepsin; 3) transport, including lactotransferrin, transferrin, ceruloplasmin (copper-binding ferroxidase), copper-containing amiloride-sensitive amine oxidase (amiloride-binding protein 1 or diamine oxidase), serum albumin, calcium-activated chloride channel regulator 1 (gob5 or calcium-activated chloride channel family member 3), zinc transporter ZIP4 (solute carrier family 39 member 4), and 24p3 (lipocalin 2).
      An abundance of immune response-related proteins detected in ULF is not surprising because a reported function of the female reproductive tract fluid is to protect against infection via the innate and adaptive immune systems (
      • Wira C.R.
      • Fahey J.V.
      • Sentman C.L.
      • Pioli P.A.
      • Shen L.
      Innate and adaptive immunity in female genital tract: cellular responses and interactions.
      ). Except for proteins directly involved in the complement pathway, peptidoglycan recognition protein and LPS-binding protein are known as pathogen-associated molecular patterns receptors, and CD14 mediates the response to LPS. Even lactotransferrin has been accorded an important role in preventing infection from pathogens in human cervical-vaginal fluid (
      • Dasari S.
      • Pereira L.
      • Reddy A.P.
      • Michaels J.E.
      • Lu X.
      • Jacob T.
      • Thomas A.
      • Rodland M.
      • Roberts Jr., C.T.
      • Gravett M.G.
      • Nagalla S.R.
      Comprehensive proteomic analysis of human cervical-vaginal fluid.
      ). Lactotransferrin is also known to interact with accessory molecules involved in the TLR4 pathway, including CD14 and LPS-binding protein, suggesting that it may activate components of the TLR4 pathway (
      • Baveye S.
      • Elass E.
      • Fernig D.G.
      • Blanquart C.
      • Mazurier J.
      • Legrand D.
      Human lactoferrin interacts with soluble CD14 and inhibits expression of endothelial adhesion molecules, E-selectin and ICAM-1, induced by the CD14-lipopolysaccharide complex.
      ,
      • Elass-Rochard E.
      • Legrand D.
      • Salmon V.
      • Roseanu A.
      • Trif M.
      • Tobias P.S.
      • Mazurier J.
      • Spik G.
      Lactoferrin inhibits the endotoxin interaction with CD14 by competition with the lipopolysaccharide-binding protein.
      ). Likewise several of the protease or antiprotease proteins identified have been detected in human uterine and cervical-vaginal fluid (
      • Dasari S.
      • Pereira L.
      • Reddy A.P.
      • Michaels J.E.
      • Lu X.
      • Jacob T.
      • Thomas A.
      • Rodland M.
      • Roberts Jr., C.T.
      • Gravett M.G.
      • Nagalla S.R.
      Comprehensive proteomic analysis of human cervical-vaginal fluid.
      ,
      • Parmar T.
      • Sachdeva G.
      • Savardekar L.
      • Katkam R.R.
      • Nimbkar-Joshi S.
      • Gadkar-Sable S.
      • Salvi V.
      • Manjramkar D.D.
      • Meherji P.
      • Puri C.P.
      Protein repertoire of human uterine fluid during the mid-secretory phase of the menstrual cycle.
      ). Among these proteases, aminopeptidase N (CD13), which is a type II metalloprotease, is involved in the trimming of antigen and the process of antigen presentation (
      • Luan Y.
      • Xu W.
      The structure and main functions of aminopeptidase N.
      ). Cathepsin B is a cysteine protease that functions in protein catabolism and may also be involved in processing of antigens in the immune response as well as hormone activation (
      • Katunuma N.
      • Matsunaga Y.
      • Himeno K.
      • Hayashi Y.
      Insights into the roles of cathepsins in antigen processing and presentation revealed by specific inhibitors.
      ). Other proteins such as laminin play a central role in the formation and stability of basement membrane, and chitinase 3-like protein is associated with down-regulating the catabolic or degradative aspects of the inflammatory response (
      • Ling H.
      • Recklies A.D.
      The chitinase 3-like protein human cartilage glycoprotein 39 inhibits cellular responses to the inflammatory cytokines interleukin-1 and tumour necrosis factor-α.
      ).
      More intriguingly is the elusive function of 24p3, a major component of ULF. The 24p3 protein has been implicated in diverse physiological processes, including apoptosis, iron transport, proinflammatory processes, and cell survival (
      • Lin H.H.
      • Li W.W.
      • Lee Y.C.
      • Chu S.T.
      Apoptosis induced by uterine 24p3 protein in endometrial carcinoma cell line.
      ,
      • Richardson D.R.
      24p3 and its receptor: dawn of a new iron age?.
      ,
      • Liu Q.
      • Nilsen-Hamilton M.
      Identification of a new acute phase protein.
      ,
      • Tong Z.
      • Wu X.
      • Ovcharenko D.
      • Zhu J.
      • Chen C.S.
      • Kehrer J.P.
      Neutrophil gelatinase-associated lipocalin as a survival factor.
      ). Notably in the context of the reproductive system, functional interactions with spermatozoa have been indirectly demonstrated through its sperm motility-enhancing and acrosome reaction-suppressing activities (
      • Lee Y.C.
      • Liao Jr., C.
      • Li P.T.
      • Tzeng W.F.
      • Chu S.T.
      Mouse lipocalin as an enhancer of spermatozoa motility.
      ,
      • Lee Y.C.
      • Elangovan N.
      • Tzeng W.F.
      • Chu S.T.
      Mouse uterine 24p3 protein as a suppressor of sperm acrosome reaction.
      ) as well as its internalization for ferric ion delivery (
      • Elangovan N.
      • Lee Y.C.
      • Tzeng W.F.
      • Chu S.T.
      Delivery of ferric ion to mouse spermatozoa is mediated by lipocalin internalization.
      ). Here we provided evidence that 24p3 could directly bind to spermatozoa and showed by sperm motility assay that co-incubation of 24p3 with oligosaccharides containing specific fucosylated terminal epitopes in the forms of Lex, Ley, and type II H, but not terminal LacNAc or 6′-sialyl LacNAc, would attenuate its enhancer activity. However, it seems that not all ULF proteins carrying the same fucosylated epitopes would bind and interact similarly. In particular, only 24p3, but not lactotransferrin, could visibly enhance the sperm motility. One hypothesis is that the relative density and multivalent presentation of the implicated glycoepitopes may be the critical determining factors. Structural modeling suggested that the N-glycosylation sites of these two proteins were similarly exposed on the surface (data not shown). However, the relatively smaller size of 24p3 may contribute to a better contact with the spermatozoa by exhibiting a higher glycan density relative to the larger lactotransferrin where the two occupied N-glycosylation sites are individually located on each of the two lobes. Another hypothesis is that not only the glycan moiety but the protein structure and conformation itself are also essential for productive interaction. To distinguish between these possibilities, further assays using recombinant 24p3 expressing no or other types of glycan structures will be helpful.
      Irrespective of the putative roles of protein carriers, the biological functions of Ley/x per se in mouse reproductive system remain unclear. Interactions with spermatozoa would implicate the presence of a specific lectin on the sperm surface that has yet to be identified. Taking a different approach, our comparative glycomics analyses of mouse reproductive luminal fluids from male to female, including EF, SVF, and ULF, clearly indicated that the expression of Ley, which requires the activity of α2-fucosyltransferase, was restricted to female ULF, whereas Lex was not found in the epididymal tract but was present in SVF and ULF. Previous study has further demonstrated that the expression of the α2-fucosyltransferase mRNA transcript in the uterine epithelium cells is dynamically regulated in concert with the estrous cycle (
      • Kimber S.J.
      • Lindenberg S.
      Hormonal control of a carbohydrate epitope involved in implantation in mice.
      ). In this work, the ULF sample was collected from 3-week-old female mice pretreated with DES, which is a synthetic estrogen that acts via the same mechanism as estrogen in stimulating the mouse uterine epithelial secretory response. DES binds to intracellular estrogen receptors with an affinity ∼100 times higher than the endogenous estrogen (
      • Chae K.
      • Lindzey J.
      • McLachlan J.A.
      • Korach K.S.
      Estrogen-dependent gene regulation by an oxidative metabolite of diethylstilbestrol, diethylstilbestrol-4′,4"-quinone.
      ). Treatment of mice with DES would markedly increase the production of ULF, thus providing a more readily available sample source for our detailed analyses. To evaluate whether this treatment truly reflected the physiological situation, we additionally collected and analyzed the ULF samples from female mice at the estrous phases of the cycle. 1D SDS-PAGE analysis showed a protein expression pattern similar to that from DES-treated sample except for less extensive complement C3 degradation, whereas Western blot analysis against Lex/y likewise indicated no obvious difference in its expression pattern (data not shown). More importantly, when comparative MS glycomics mapping was performed for the N-glycans derived from different stages of the estrous cycle, a high expression level of Lex/y epitopes was only detected during the estrous phase with a much a lower level during proestrous phase and a level that was hardly detectable during metestrous and diestrous phases (data not shown).
      Thus, both Lex/y and 24p3 expression levels in ULF are elevated during the estradiol surge in proestrous and estrous phases and suppressed when progesterone rises to a high level at metestrous and diestrous phases. Its sperm-binding activity and motility-enhancing properties therefore correlate with the fertile window of mouse estrous cycle. This is in contrast to human glycodelin, which carries Lex/y only in the glycoform found in the male seminal plasma but not that from female uterine origin (
      • Seppala M.
      • Taylor R.N.
      • Koistinen H.
      • Koistinen R.
      • Milgrom E.
      Glycodelin: a major lipocalin protein of the reproductive axis with diverse actions in cell recognition and differentiation.
      ). The expression of the latter glycoform was found to be at a minimum during the periovulatory midcycle but increased significantly from the 4th postovulatory day and contributed to the contraceptive activity in the latter half of the secretory phase of the menstrual cycle. Thus, hormone-regulated, gender-specific glycosylation is a common feature for human and mouse reproductive tracts. Both systems involve Lex/y specifically but in a different context. A unifying model for functional roles at the molecular level is still lacking at present but would surely benefit from current glycomics and glycoproteomics studies.

      Acknowledgments

      LTQ-Orbitrap data were acquired at the Academia Sinica common mass spectrometry facilities located at the Institute of Biological Chemistry. We are grateful to He-Hsuan Hsiao for technical assistance in the early stage of this work and Dr. Han-Jia Lin for providing the SVF samples for analysis.

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