Placenta (1991), 12, 637-651
Syncytiotrophoblast Membrane Protein Glycosylation Patterns in Normal Human Pregnancy and Changes with Gestational Age and Parturition P. D. ARKWRIGHTa~b~c, C. W. G. REDMAN”, P. J. WILLIAMSb, R. A. DWEK” & T. W. RADEMACHERb a Nufield Department of Obstetrics,John Radcltfe Hospital, Headington, Oxford, OX3 9DU, UK b Glycobiology Unit, Department of Biochemistry, lJniversi(y of Oxford, South Parks Rd, Oxford, OXI 3QlJ, UK ’To whom correspondence should be addressed Paper accepted 17.0.5.1991
INTRODUCTION The surface membrane of human fetally derived syncytiotrophoblast, lining the intervillous spaces, makes primary contact with the maternal circulation over its 10 m2 interface, and may therefore influence many interactions between the trophoblast and maternal blood. Cell surface oligosaccharides contribute to the structure of the glycocalyx, the outermost cell coat of most, if not all plasma membranes, and may thus influence a variety of cellular interactions. The N-linked oligosaccharides of glycoproteins range from relatively unprocessed oligomannose structures, to highly processed multi-antennary sialylated complex structures (Robbins et al, 1977; Lennarz, 1980). Spontaneous or experimentally induced changes in the expression of N-linked oligosaccharides on the cell surface can result in alterations in cell behaviour, depending on the physiological timing and environment. For example, an increase in terminal mannose [as on oligomannose structures, common to many primitive organisms such as yeast and fungi (Kornfeld & Kornfeld, 1980)], or terminal N-acetylglucosamine (GlcNAc) (which is uncommon, but the result of incomplete processing of complex structures) on the cell surface, may promote cell growth and organogenesis in the developing embryo (Shur & Hall, 1982; Bayna et al, 1986; Begovac & Shur, 1990), or cell death by activation of the immune system, and removal of cellular debris (Sharon, 1984; Duvall et al, 1985). Cells expressing high densities of these oligosaccharides are more sensitive to natural killer cell lysis than cells expressing completely processed sialylated complex-type oligosacchar-ides (Ahrens & Ankel, 1987; 1988). Changes in cell-surface oligosaccharides are also a 0143-4004/91/060637 + 15 $05.00/O
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common finding in tumor cells, and particular linkages and residues are associated with tumorigenic and metastatic phenotypes, specifically, there may be an elevation of the /31-6 branching enzyme (Dennis et al, 1987). This increased branching may be associated with an increased sialylation. One of the most important functions of the syncytiotrophoblast in the placenta is in replacing the endothelium, to maintain a surface which promotes blood flow and active interchange of plasma solutes. Its integrity, and that of the intervillous circulation must depend on its ability not to stimulate inappropriately such blood cells as platelets (to avoid thrombosis), or immune cells (to avoid immune rejection). Although both functions are likely to be complex, it is to be expected that the structure of the glycocalyx, of which the plasma membrane N-linked oligosaccharides are one part, would contribute. Recent evidence confirms this, and has established the critical importance of specific cell surface carbohydrate epitopes (Lewis X antigens) in determining interactions between endothelium and both platelets and leukocytes (Springer & Lasky, 1991; Larsen et al, 1990; Lowe et al, 1990). Antibody binding studies suggest that the syncytiotrophoblast does not usually express the Lewis antigen although other trophoblast subpopulations may express it (King & Loke, 1988). Further direct structural studies which examine the temporal changes of trophoblast membrane oligosaccharides during normal and abnormal pregnancy are indicated. In this study, the N-linked oligosaccharide structures of the surface membrane proteins of the syncytiotrophoblast are characterized in detail for the first time, and the temporal changes which occur during gestation and parturition are determined, as a first step in delineating their possible functions, hinted at above.
MATERIALS
AND METHODS
Collection of placentae Placentae were collected from women within 2 h of delivery. Spontaneous labour was defined as the initiation of labour, and vaginal delivery, without the use of drugs or surgical intervention. In women who had premature labours and deliveries, spontaneous labour was not attributable to congenital fetal abnormality, maternal infections or other illnesses. In women who were induced, labour was initiated with a vaginal 3 mg PGEz pessary, followed by artificial rupture of membranes, and in some cases intravenous oxytocin infusion. All these women delivered vaginalIy. Women who had caesarean sections, did not go into labour, and the indications for elective section were cephalopelvic disproportion, abnormal fetal lie, or a previous caesarean section. Gestational age of all women was determined from the history of the last menstrual period, and ultrasound scan performed in the first trimester. Isolation of syncytiotrophoblast membrane glycoproteins
An extract of purified syncytiotrophoblast membrane microvilli was prepared using the method of Smith et al (1974). Two-thirds of the thickness of the placental cotyledons were dissected from the overlying maternal decidua, and washed with 2 L of 50 mu CaC12 followed by 500 ml ofphosphate-buffered saline. The pieces were then minced, and added to 350 ml of 0.15 M NaCl, pH 7.4, and stirred for 60 min at 4°C. After fihering through a nylon sieve, the filtrate was centrifuged at 800 g for 10 min. The supernatant was centrifuged at 10000 g for 10 min to remove large ceil&r debris. The resuhant supematant was centrifuged at 100 OOOgfor 45 min. The pellet containing the syncytiotrophoblast mem-
F&w-e 1. Electron micrographs of term syncytiotrophoblast. (a) Placental villi before processing. (b) Placental villi after processing to remove microvilli (method of Smith et al, 1974). (c) Purified syncytiotiophoblast microvilli.
brane microvilli (STB) was collected and stored at -20°C until required. The validity of the method was confirmed by electron microscopy of the syncytiotrophoblast cells before and after extraction of the microvilli, and of the microvillous preparation (Figure 1). Figure 1 (b) shows that the extraction method sheared the majority of the microvilli from the syncytial surface without distruption of the underlying cellular architecture. Extrinsic proteins were isolated from the STB pellet by extraction with 3 M KC1 for 24 h at 4°C (Khalfoun et al, 1986). Insoluble material was sedimented by centrifugation at 100 OOOg for 45 min. The supernatant was dialysed against repeated changes of distilled water for 48 h. Total proteins were isolated from the STB pellet with acetone (20 ml/g pellet) at -20°C. The protein was pelleted by centrifugation at 500 g for 10 min, before excess acetone was ea-aporated with Na, and was dialysed with repeated changes of distilled water for 48 h. Preparation of radiolabelled oligosaccharide libraries from the glycoprotein extracts To prepare libraries of protein-linked oligosaccharides from the microvillous membrane, 2 mg aliquots of the lyophilized salt-free glycoprotein extracts were cryogenically dried over activated charcoal at - 196°C (< 1 0p4 bar). The extract was then dissolved in freshly doubledistilled anhydrous hydrazine (0.1 ml/mg protein; toluene/CaO, 25°C 10 Torr) under an anhydrous argon atmosphere (Ashford et al, 1987; Parekh, 1985). The temperature was raised 17Wh from 30 to 85°C and then maintained at 85°C for a further 12 h with shaking. The hydrazine was removed by evaporation under reduced pressure (