The Carbohydrate Structure of Porcine Uteroferrin and the Role of the High Mannose Chains in Promoting Uptake by the Reticuloendothelial Cells of the Fetal Liver*

Uteroferrin, the iron-containing, progesterone-in- duced phosphatase of the porcine uterus, is a glycoprotein carrying a single oligosaccharide chain. Most of the uteroferrin isolated from either uterine secretions or allantoic fluid has endoglycosidase H-sensitive car- bohydrate chains with either five or six mannose residues. As determined by ‘H-NMR spectroscopy, the Mane oligosaccharide has the following structure.

isolated from either ovalbumin or uteroferrin. Rat cells did not accumulate uteroferrin whose high mannose chains had been removed using endoglycosidase H. Moreover, the K uptake values (3 X 10" M), specific competition by D-mannOSe and L-fucose bovine serum albumin, and inhibition by EDTA are consistent with an uptake mechanism involving a receptor for highmannose oligosaccharides on the liver sinusoidal cells. It is suggested that one function of this receptor in the fetal pig is to remove maternally derived uterine glycoproteins from the fetal circulation. In the case of uteroferrin this process provides iron to the fetal liver.
Uteroferrin (Uf') is a purple-colored glycoprotein with acid phosphatase activity (1, 2) which is synthesised by the glandular endometrium of pigs (3). Its production is under the control of progesterone (4) and it is a major secretory product of the midpregnant uterus (1). The purple coloration of Uf results from an iron center in which the metal is coordinated to one or more tyrosine residues (5, 6). There is some controversy about the iron content of different preparations of Uf, but up to two atoms can be bound (see Ref. 7). A considerable body of evidence has accumulated to suggest that a major function of Uf is the transplacental transport of iron during pregnancy (1,3,8,9). In the pig, placentation is of the diffuse epitheliochorial type (10) with several cell layers separating maternal and fetal blood supplies, an arrangement which is believed to require an indirect transfer of iron from mother to conceptus (9,11). Special placental (chorionic) structures, known as areolae, develop opposite the mouths of uterine glands and appear to be involved in the uptake of secreted proteins (12). Uf is the major iron-containing component of porcine uterine secretions (1) and can be detected in areolae as well as in the placental venous drainage (3). The known major sites of Uf metabolism in the conceptus are allantoic fluid (8,13) and liver (8,9). The Uf present in allantoic fluid is thought to represent excess protein not immediately cleared from the fetal blood by the liver (3). Immunocytochemical studies have suggested that Uf forms part of the urinary filtrate and enters allantoic fluid via the bladder and urachus (3). Once there it is rapidly broken down and loses its iron to fetal transferrin (8). The liver, however, is the major site of erythropoiesis and iron metabolism in the fetal pig at midpregnancy (14). Because many glycoproteins introduced into the blood stream of mammals are cleared by the liver by a mechanism that involves surface receptors with lectin-like specificities (15), we have examined whether the clearance of Uf from fetal blood might involve its carbohydrate. An initial analysis of Uf indicated that the majority of the molecules lacked sialic acid, but contained glucosamine, mannose, and some galactose (4). However, the presence of glucose was also reported, suggesting that some of the preparations were contaminated with exogenous carbohydrate. In this study we have re-evaluated the oligosaccharide structure of Uf and examined the role of carbohydrate in mediating the binding and retention of this glycoprotein in the liver.

DISCUSSION
The results in this paper demonstrate that the purple protein, Uf, isolated from either uterine secretions or from allantoic fluid, is a glycoprotein containing about 4.8% by weight carbohydrate. This carbohydrate is present on single oligosaccharide chains and consists mainly of high mannose structures which could be released by Endo H and which bound strongly to ConA-Sepharose. HPLC analysis revealed that the major oligosaccharides released by Endo H had the empirical formulae Man5GlcNAc and Man&lcNAc. The structures of these oligosaccharides were determined by means of 'H-NMR analysis and are shown in Table 11. Results obtained using al,2-mannosidase to test for the presence of al,Z-linked, terminal mannosyl residues on the purified Mans and Man5 oligosaccharides were entirely consistent with the assignments in Table 11, i.e. a single residue of mannose was released from the Man6 species but not from the Man5.
A small proportion of Uf carbohydrate appeared to be in the form of complex or hybrid-type chains as evidenced by the presence of trace amounts of galactose and sialic acid (Table I) and by the fact that a small fraction bound either weakly or failed to bind to ConA. In addition, about 5% bound to wheat germ agglutinin-Sepharose. Uf can also be labeled by the galactose oxidase-NaB3H4 technique: a procedure which depends upon the availability of galactosyl residues in the carbohydrate (16).
Only a very small proportion of the Uf molecules purified from allantoic fluid carried phosphorylated oligosaccharide chains. This phosphate was presumed to exist as mannose 6phosphate since it is in this form that it is found on Uf released from cultures of uterine endometrium (17). A high proportion (up to 30%) of newly synthesized Uf carries this group, although it is masked by a covering N-acetylglucosamine residue (17). The oligosaccharide chains of newly synthesized Uf are also larger than those of the mature Uf studied here (17). We have suggested that the Oligosaccharide chains on Uf continue to be modified following their release into the uterine lumen so that they progressively lose phosphate and outer saccharide residues and thus become smaller in size (17). Extensive metabolism of Uf iron occurs in the liver of the midpregnant fetal pig (8,9). As demonstrated here, lz5Tlabeled Uf introduced into the umbilical vein is taken up almost exclusively by the cells lining the liver sinusoids and not by parenchymal cells (Fig. 6). This population consists of the Kupffer and endothelial cells. In our study we have not been able to determine whether one or both of these cell types are involved in Uf uptake, and we have classified them together as reticuloendothelial cells. Uptake of lZ5I-labeled Uf, both in vivo and in vitro, was blocked by addition of unlabeled Uf. Although a statistical analysis was not carried out, autoradiographic studies (Fig. 5 B ) strongly suggested that the labeled Uf became internalized rapidly into endocytic vacuoles. Together, these experiments indicated that Uf was taken up by reticuloendothelial cells by a receptor-mediated process.
Within the past 10 years a considerable body of information has accumulated concerning the uptake of glycoproteins by mammalian cells. Of particular relevance to this study is the presence in the liver of distinct populations of receptors that bind specifically to particular carbohydrate groups (for review see Ref. 15). A receptor which binds certain oligosaccharides terminating in D-mannose, t-fucose, or N-acetylglucosamine is found on reticuloendothelial cells of the liver (18-21) and on macrophages (22-24). Binding is characteristically inhibited by yeast mannan and by glycoproteins terminating in amannosyl groups. Since binding appears to require calcium, EDTA is also a potent inhibitor. Our results, using partially purified populations of reticuloendothelial cells from adult rat and fetal pig livers, suggest that the uptake of Uf is mediated by a mannose-specific cell surface receptor. For example, Uf binding to rat cells was inhibited by EDTA, by high mannose glycoproteins, and glycopeptides (Table V and Fig. 7) and by bovine serum albumin substituted with D-mannOSe or Lfucose residues but not with D-galaCtOSe (Table V). The kinetic constant for uptake of Uf (approximately 3 X M) ( Fig. 8) was very close to that noted for uptake of oligosaccharides terminating in mannose by rat reticuloendothelial cells (19). Although fewer experiments were carried out with equivalent cells from the fetal pig liver (Table VI), the characteristics o f Uf uptake appeared to be identical to those seen with the rat cells. Therefore, even though a detailed study of the characteristics of the uptake process was not carried out, the results are entirely consistent with the hypothesis that the high mannose receptor, previously described on macrophage (22-24) and reticuloendothelial cells (18-21), mediates uteroferrin uptake by the fetal pig liver.
Binding of Uf to crude membrane fractions from fetal pig livers has also been demonstrated (3). Results from more recent experiments are consistent with the view that such binding occurs to a receptor that recognizes high mannose oligosaccharide chains and has a K D for Uf of about 2.8 x M: However, we have not pursued these studies in detail since it was impossible in our studies to determine the origin of the receptors, i.e. the cell type involved, and whether the receptors were located at the cell surface or on internal membrane systems (see Refs. 15 and 23). Their relevance in Uf uptake was therefore unclear.
The fate of Uf, once it has been taken up by the liver cells, is not known. The protein has long been believed to play partial and possibly a major role in supplying iron to the fetus until at least day 75 or so of pregnancy (1,8,9,25). It is certainly synthesized during this period in amounts adequate for the requirements of fetal hematopoiesis, and its iron is readily incorporated into fetal hemoglobin (8)

Structure and Function of Uteroferrin Carbohydrate
is synthesized in the liver. How Uf iron reaches the developing blood cells within the blood islands is unclear. Uptake of Uf by the reticuloendothelial cells appeared to involve coated pits and presumably resulted in internalization into an endosome compartment which was likely to have an acid pH (see Refs. 26 and 27). It had probably not entered lysosomes because of the short interval from infusion of lZ5Ilabeled Uf and tissue fixation, i.e. 3-4 min. This time interval is probably insufficient for '251-Uf taken up by endocytosis to be transported to lysosomes. For example, a2-macroglobulin was not detected in lysosomes until 15-30 min after exposure of fibroblast(s) to the ligand (28). A 15-min time interval was also required between exposure of rat hepatic sinusoidal cells to '251-glycoproteins with terminal mannose and N-acetylglucosamine residues and the detection of these proteins in lysosomes (29).
Unlike transferrin (30-33), Uf binds its iron tightly down to pH 3 (8) and would not be expected to release its iron as a result of the low pH within the endosome or lysosome. However, while the transferrin receptor continues to bind apotransferrin at around pH 5 (30, 31), the mannose receptor is believed to release its ligand (see Refs. 15 and 23). Thus Uf internalized on the mannose receptor would not be returned to the cell surface, as is transferrin (30-33). Rather, it would most probably move into lysosomes. It is possibly at this location that Uf is degraded and its iron is released. Alternatively, Uf may be transferred in intact form from the reticuloendothelial cells to neighboring blood islands.
In conclusion, these experiments strongly suggest that the high mannose oligosaccharides of Uf function in targeting the molecule to the fetal liver where its iron is used for erythropoiesis. Whether its carbohydrate also plays a role in uptake of Uf by the placenta, and movement of the glycoprotein in intact form across the chorionic epithelium into the placental blood capillaries, remains to be determined.  -

Addition of unlabeled Uf depressed uptake by abouc 672 by the end of 1 h. Y e a s t maonan and ovalbumin also inhibited the accumvlation of ['2511-lebeled-Uf by the cells.
In incubations carried out at 3 7 V , cell associated radioncrivicy with high mannose-type chains purified by chromatography on Con A Uvtake of intact and aglyco-Uf. Uf was radioiodinated and wlecules Sepharose. A portion of this material was treated with endo H. sod, after passage of the digest through a second Con A affinity colum, rhe unbound H-treated Uf by rat reticuloendothelial cells during a 1 h incubation. fraction collected. Table 4 compares the uptake of the intact and endo  Inhibitors Of Uf uptake. The result. of several different cxparimente using a variety of potential inhibitors and ccmpeting ligand. for uptake of Uf by rat liver reticuloendothelial cell. are reported io Table 5.