(Received for publication, May 16, 1995; and in revised form, August 2, 1995)
From the
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:
Gal1-4GlcNAc (lacNAc), GalNAc
1-4GlcNAc
(lacdiNAc), NeuAc
2-6Gal
1-4GlcNAc (sialylated
lacNAc), NeuAc
2-6GalNAc
1-4GlcNAc (sialylated
lacdiNAc), Gal
1-4(Fuc
1-3)GlcNAc
(Lewis
), and GalNAc
1-4(Fuc
1-3)GlcNAc
(lacdiNAc analogue of Lewis
). 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.
Bohn and co-workers originally isolated a glycoprotein from
human placenta that they designated placental protein 14 or PP14 ()(Bohn et al., 1982). PP14 was subsequently found
to be synthesized not by the placenta but by the secretory and
decidualized endometrium (Julkunen et al., 1986a, 1988). PP14
was therefore also referred to as progesterone-associated endometrial
protein or PAEP in accordance with its endometrial origin. More recent
evidence indicates that PAEP is also synthesized by the hematopoietic
tissues of the bone marrow (Kamarainen et al., 1994) and
perhaps other tissues. Since the glycoprotein referred to as PP14 or
PAEP is not of placental origin nor is it exclusively synthesized in
the endometrium, previous designations may not truly reflect its
diverse sites of synthesis or its function. Therefore in this paper, we
have designated PP14/PAEP as ``glycodelin'' to eliminate
confusion over these issues and to emphasize the unique nature of
oligosaccharides in this glycoprotein.
The temporal and spatial expression of glycodelin in the reproductive organs of the human female is highly regulated. During the menstrual cycle, glycodelin is not expressed in the proliferative endometrium but increases significantly from the fourth postovulatory day, peaking around the 12th day (Julkunen et al., 1986a). Thus glycodelin expression is at a minimum during the peri-ovulatory period of the cycle. However, at the time of implantation of the embryo, glycodelin synthesis in the decidua is induced to very high levels (4-10% of total protein) (Julkunen et al., 1985). Glycodelin is also secreted into the amniotic fluid in substantial amounts, reaching a peak in the 10th to 14th week of gestation (Julkunen et al., 1985). In addition, glycodelin is also found in the serum under normal conditions and during pregnancy, although at a much lower level than in amniotic fluid or decidual cells (Julkunen et al., 1986b).
Glycodelin manifests several significant biological activities when tested in immunological assay systems. Crude decidual extracts containing this glycoprotein were initially shown to suppress thymidine uptake in both normal and mitogen-stimulated human mixed lymphocyte culture (Bolton et al., 1987; Pockley et al., 1988). Decidual extracts containing glycodelin also decreased the synthesis of cytokines (interleukin-1 and interleukin-2) and interleukin-2 receptors by mitogen-stimulated cells (Pockley and Bolton, 1989, 1990). Purified glycodelin also suppresses the lysis of K562 cells by human natural killer cells at low concentrations (Okamoto et al., 1991). Therefore, glycodelin may be one of several factors that induce highly regiospecific immunosuppression of the maternal response to the human embryo/fetus. Another significant biological activity of glycodelin is its ability to inhibit human sperm-zona pellucida binding in the hemizona assay system (Oehninger et al., 1995). To date, glycodelin is the most potent glycoprotein inhibitor of human sperm-zona pellucida binding in this assay system.
We have recently
proposed that human sperm-zona pellucida binding requires a
selectin-like interaction between human sperm and human zona pellucida
(Patankar et al., 1993a, 1993b). Therefore, we hypothesized
that glycodelin probably manifested its immunosuppressive and
contraceptive activities via its oligosaccharide chains. Previous work
has indicated that this glycoprotein contains 17.5% carbohydrate by
weight (Bohn et al., 1982). However, no oligosaccharide
structures have been reported. In this study, we have performed
structural analysis of glycodelin-derived N-linked
oligosaccharides and glycopeptides using mapping strategies (Morris et al., 1983, Dell et al., 1983) based upon fast atom
bombardment (FAB) and electrospray (ES) mass spectrometry. Glycodelin
has three consensus sites for N-linked glycosylation (Julkunen et al., 1988) (Fig. 1), and we have shown that the
first two of these sites are glycosylated with defined and
substantially different heterogeneous populations of glycans. Many of
these glycans have antennae composed of sialylated or fucosylated
GalNAc1-4GlcNAc(lacdiNAc) sequences, which are rare in
higher animals. N-Linked oligosaccharides of this type have
been shown to be potent inhibitors of selectin-mediated adhesions
(Grinnell et al., 1994), consistent with our hypothesis that
glycodelin blocks both human sperm-zona pellucida binding and immune
cell function via its oligosaccharide chains.
Figure 1: Amino acid sequence of human glycodelin (Julkunen et al., 1988). Underlined regions represent consensus sequences for N-glycosylation.
A unique feature of mass spectrometry, namely the ability to derive definitive structural information from mixtures (in contrast to other spectroscopies normally requiring pure samples for study), was recognized and exploited by us in specifically designed strategies for ``mixture analysis'' some years ago (Geddes et al., 1969; Morris et al., 1971; Morris et al., 1978). The masses of component peaks alone (even in the absence of fragmentation) are diagnostic not only for the composition of biopolymers but also for sequence, by relating the masses observed to biosynthetic pathways (oligosaccharides) or amino acid/cDNA-derived sequences (oligopeptides), leading to the concept of mapping biopolymer structures by mass spectrometry (Lemaire et al., 1982; Dell et al., 1983; Morris et al., 1983). These strategies have been applied here to glycodelin to map and differentiate the glycopeptides from proteolytic digests and to define and locate glycosylation in the molecule.
Figure 2: FAB mass spectrum of permethylated N-glycans from glycodelin: molecular ion region (a) and fragment ion region (b). N-Glycans were released from glycodelin tryptic glycopeptides by digestion with PNGase F, isolated by Sep-Pak, and permethylated.
Figure 3: UV chromatogram (upper trace) and TIC (lower trace) of glycodelin peptides and glycopeptides analyzed by on-line microbore LC-ES-MS.
Figure 4: Transformed electrospray mass spectrum of the glycopeptides spanning Asn-28 of glycodelin. Glycodelin was digested overnight with CNBr. The dried sample was then reduced with dithiothreitol in triethylamine and dried again. The sample was analyzed by LC-ES-MS.
Figure 5: FAB mass spectrum of permethylated released N-glycans from Asn-28 of glycodelin. The LC-ES-MS fraction containing the Asn-28 glycopeptide was digested with PNGase F. N-Glycans were isolated by Sep-Pak and the void fraction dried and permethylated.
Combining scans 139-145 produces the raw data (multiply charged) shown in Fig. 6. The complex appearance of this spectrum, which contrasts with the clean single signals of different charge states expected for a peptide, is immediately indicative of the expected heterogeneity seen in a glycopeptide. Computing the charge states shown, and transformation of the data produces component masses of 11,835.5, 11,879.3, and 12,126.8 for the most abundant peaks. These masses are approximately 2000 Da higher than the anticipated mass of peptide Ala-33 to Met-117, indicating glycosylation of the peptide. The peptide contains potential glycosylation sites Asn-63 and Asn-85, but since we have already proven that Asn-85 is not glycosylated (see earlier FAB and ES mapping experiments), it follows that the glycans on peptide Ala-33 to Met-117 are attached to Asn-63. Their identities were studied in detail by FAB-MS analysis after their release by PNGase F from the glycopeptides in collected fractions 56-57 (see Fig. 7and Table 7). It is noteworthy that the majority of glycans in this sample are different from those attached at Asn-28 (see below).
Figure 6: ES-MS spectrum of glycodelin glycopeptides spanning Asn-63 and Asn-85. The bracketed numbers show the charge states of the ions.
Figure 7: FAB mass spectrum of permethylated released N-glycans from Asn-63 of glycodelin. The LC-ES-MS fraction containing the Asn-63 glycopeptide was digested with PNGase F. N-Glycans were isolated by Sep-Pak and the void fraction dried and permethylated.
Figure 8: Structures of the major N-glycans present at Asn-28 (a) and Asn-63 (b) of glycodelin. Panel a, superscript a indicates that minor forms may exist with different arm structures as indicated by presence of 3- and 6-linked mannose; superscript b indicates that fucose residue may be 3-linked to the GlcNAc on either arm. Panel b, superscript c indicates that the fucose residue may be 3-linked to the GlcNAc on either arm, but is not on the arm bearing the sialic acid.
The major non-reducing epitopes
in the glycodelin complex-type glycans are: (i) Gal1-4GlcNAc
(lacNAc), (ii) GalNAc
1-4GlcNAc (lacdiNAc), (iii)
NeuAc
2-6Gal
1-4GlcNAc (sialylated lacNAc), (iv)
NeuAc
2-6GalNAc
1-4GlcNAc (sialylated lacdiNAc),
(v) Gal
1-4(Fuc
1-3)GlcNAc (Lewis
), and
(vi) GalNAc
1-4(Fuc
1-3)GlcNAc (the lacdiNAc
analogue of Lewis
). The relative abundances of molecular
ions in the ES and FAB spectra indicated that lacNAc- and
lacdiNAc-containing epitopes are of comparable abundance and that
approximately 60% of the glycans are sialylated and about 20% of the
glycans have fucosylated antennae. It is notable that about 30% of the
biantennary glycans bear lacNAc and lacdiNAc antennae within a single
structure. Additional quantitative information was obtained from sugar
analysis of trimethylsilyl ether methyl glycosides of the total glycan
population (data not shown). These experiments gave a Gal:GalNAc ratio
(translating into a lacNAc:lacdiNAc ratio) of 1.2:1, which supports the
conclusions from the MS data.
The majority of the glycodelin N-linked oligosaccharides characterized in this study are not typically found in mammalian glycoproteins. In particular, the presence of lacdiNAc-containing antennae is unusual because, with the exception of the pituitary glycohormones, this sequence has been rarely observed in the glycoproteins of higher animals (see Dell and Khoo(1993), van den Eijnden et al.(1995), and references cited therein).
The
best characterized family of mammalian lacdiNAc glycoproteins are the
pituitary glycohormones, which contain sulfated lacdiNAc structures
(Baenziger and Green, 1988). The GalNAc transferase, which adds GalNAc
to these glycoproteins, recognizes the tripeptide motif Pro-Xaa-Arg/Lys
(PXR/K) located 6-9 residues NH-terminal to
an Asn glycosylation site (Smith and Baenziger, 1992). This GalNAc
transferase, together with the sulfotransferase responsible for
synthesizing the unique sulfated epitope on the pituitary
glycohormones, is present in a number of tissues other than the
pituitary, and the two enzymes appear to be co-ordinately expressed
(Dharmesh et al., 1993). However, we consider it unlikely that
glycodelin is a substrate for the PXR/K-specific GalNAc
transferase because it does not contain a recognition motif 6-9
residues upstream of either glycosylation site. Furthermore, we were
not able to detect sulfated structures in glycodelin using
acetylation/FAB-MS strategies, which are optimized for the detection of
sulfated oligosaccharides (data not shown) (Khoo et al.,
1993).
Non-sulfated lacdiNAc structures of the type present in
glycodelin have previously been found in a three categories of
mammalian glycoproteins. The first comprises glycoproteins produced by
bovine mammary glands, including lactotransferrin (Coddeville et
al., 1992), CD36 (Nakata et al., 1993), and butyrophilin
(Sato et al., 1995). The second contains three human
glycoproteins, all of which are serine proteases with important
physiological functions, namely Bowes melanoma tissue plasminogen
activator (Chan et al., 1991) and urinary type plasminogen
activator (urokinase) (Bergwerff et al., 1992), both of which
convert plasminogen to plasmin, and urinary kallidinogenase (Tomiya et al., 1993), which cleaves kininogens to liberate
lysyl-bradykinin, a vasoactive peptide. The third category contains
only a single glycoprotein at present, namely human recombinant Protein
C (rHPC) expressed in human kidney 293 cells (Yan et al.,
1993), but we anticipate the discovery of many more examples with the
increasing use of this human cell line for the expression of
recombinant glycoproteins. Due to its availability in large quantities,
rHPC is among the best characterized of the mammalian
lacdiNAc-containing glycoproteins. Like glycodelin, rHPC carries a
heterogeneous population of complex-type oligosaccharides composed of
lacNAc and lacdiNAc building blocks, which are substituted with either
sialic acid or fucose (Yan et al., 1993). Interestingly, the
sialylated lacNAc antennae in rHPC have both 2-3 and
2-6 linked sialic acid, but the the former linkage was not
observed in the lacdiNAc antennae. It is notable that
NeuAc
2-3GalNAc
1-4GlcNAc has not, to our
knowledge, been found in mammalian glycoproteins, although this
structure has been identified in serine proteases derived from snake
venoms (Pfeiffer et al., 1992; Lochnit and Geyer, 1995). It is
possible that, by analogy with PXR/K-specific GalNAc
transferase and sulfotransferase (Dharmesh et al., 1993),
there could be co-ordinate expression of GalNAc transferase and
2-6-sialyltransferase in mammalian cell lines that
synthesize sialylated lacdiNAc structures.
Several lines of evidence
indicate that oligosaccharides are essential recognition sequences in
cell-mediated adhesions in both inflammatory and immune responses
(Phillips et al., 1990; Springer, 1990; Lasky, 1992;
Bevilacqua, 1993). Oligosaccharides terminated with sialylated or
sulfated Lewis type sequences have been shown to act as
specific ligands for selectin-mediated adhesions (Berg et al.,
1991; Yuen et al., 1992). Other oligosaccharide sequences can,
however, act as selectin ligands (Varki, 1994). Importantly, in the
rHPC study (see above) it was shown that a biantennary N-linked oligosaccharide bearing
GalNAc
1-4(Fuc
1-3)GlcNAc antennae is a potent
inhibitor of E-selectin-mediated adhesion (Grinnell et al.,
1994). Since the same fucosylated epitope is also expressed on
glycodelin, it is possible that a component of the immunosuppressive
effect exhibited by glycodelin is mediated via blocking of the
selectin-like binding sites by this carbohydrate sequence.
Other
specific antennae associated with glycodelin may also interact with
alternative bioactive receptor proteins of the human immune system.
CD22 is a B cell-associated receptor of the immunoglobulin superfamily
that acts as both an adhesion molecule and an activation molecule
(Clark and Lane, 1991; Clark, 1993; Ledbetter et al., 1993;
Peaker, 1994). Transfected cells that stably express CD22 on their
surfaces show greatly enhanced binding to T and B lymphocytes (Wilson et al., 1991). CD22 is closely associated with the subset of
responsive B lymphocytes as defined by stimulation with anti-µ
(Pezzutto et al., 1988). CD22 also binds to CD45, the
leukocyte-specific receptor-linked phosphotyrosine phosphatase involved
in T-cell activation (Stamenkovic et al., 1991). Previous
studies have revealed that CD22 binds to
NeuAc2-6Gal
1-4GlcNAc sequences (Powell and Varki,
1994). More recent studies indicate that CD22 also binds the
NeuAc
2-6GalNAc disaccharide with approximately equal
affinity as it does the NeuAc
2-6Gal
1-4GlcNAc
sequences (Powell et al., 1995). Therefore we believe that
glycodelin may bind to CD22 via its
NeuAc
2-6Gal
1-4GlcNAc and/or
NeuAc
2-6GalNAc
1-4GlcNAc antennae and may inhibit
specific immune cell adhesion and activation events mediated via this
receptor protein.
We also find it significant that glycodelin has
glycoforms carrying NeuAc2-6GalNAc
1-4GlcNAc and
GalNAc
1-4(Fuc
1-3)GlcNAc antennae on a single
biantennary oligosaccharide (structure xviii; Fig. 8). Although
this structure has been previously observed in rHPC (Yan et
al., 1993), we are now the first to demonstrate its expression in
a naturally occurring glycoprotein. The biological activities expressed
by this glycan remain to be determined. It is possible that this
oligosaccharide could interact with the selectins or other adhesion
molecules with selectin-like specificity via the fucosylated antenna
whereas its sialylated antenna could bind to CD22. Such an
oligosaccharide could manifest multiple biological effects, including
blocking inflammatory responses, attenuating CD22-dependent immune
responses or perhaps inhibiting other selectin-like adhesion processes.
It is also possible that certain carbohydrate-binding proteins
associated with either the immune or reproductive systems may require
the precise spatial arrangement of fucose and sialic acid provided by
the antennae for optimal binding.
Evidence collected from diverse species in both the plant and animal kingdoms indicates that the appropriate recognition of surface carbohydrates is a crucial event in the binding of sperm to the eggs during fertilization (Macek and Shur, 1988; Miller and Ax, 1990; Wassarman, 1990). In the mouse, oligosaccharides associated with the zona pellucida glycoprotein ZP3 have been shown to be recognized by specific egg-binding proteins located on the sperm plasma membrane (Wassarman, 1990). It is probable that a similar paradigm is utilized in the human system.
We have
previously suggested that initial human sperm-zona pellucida binding
involves a selectin-like adhesion (Patankar et al., 1993a,
1993b). This proposed specificity was initially based upon our
observation that fucoidan blocked initial human sperm-zona pellucida
binding (Oehninger et al., 1990) and a selectin-mediated
adhesion process (lymphocyte homing) in the same concentration range
(Yednock and Rosen, 1989). Fucoidan also blocked induction of the
sperm's acrosome reaction by solubilized human zona pellucida,
consistent with its ability to block sperm-zona pellucida binding
(Mahony et al., 1991). We recently reported in a preliminary
study that sialyl-Lewis oligosaccharide and human
orosomucoid also inhibit initial human sperm-zona pellucida binding in
the same concentration-dependent manner as is observed for
E-selectin-mediated adhesion (Clark et al., 1995a). Although
these studies suggest that the egg-binding protein is a selectin, our
preliminary studies using specific anti-selectin monoclonal antibodies
indicate that these adhesion proteins are not expressed on human sperm
(Clark et al., 1995b). Therefore, we have hypothesized that
the human egg-binding protein, though not itself a selectin, may have
converged with the selectins in its carbohydrate binding specificity.
Glycodelin also inhibits initial human sperm-zona pellucida binding in
a potent concentration-dependent manner (Oehninger et al.,
1995). The expression of putative selectin ligands on this glycoprotein
provides further evidence supporting our hypothesis that initial human
sperm-zona pellucida binding is dependent upon a selectin-like adhesion
process.
The structural studies reported in this paper provide the necessary foundation for effectively addressing the above issues. We are now investigating the potential contribution of the variety of glycans attached to glycodelin to its immunosuppressive and contraceptive activities. Finally, since glycodelin is expressed in bone marrow (Kamarainen et al., 1994; Morrow et al., 1994) and perhaps other tissues, it will be of great interest to see whether their glycosylation and function are the same. Until that information is available we propose to designate this glycoprotein isolated from amniotic fluid ``glycodelin-A.''