A critical element of lutropin bioactivity in vivo is its rapid removal from the blood by a receptor, located in hepatic endothelial cells, that recognizes the terminal sulfated carbohydrate structure SO 4 -4-GalNAc␤1,4Glc-NAc␤1,2Man␣ (S4GGnM). We have previously shown that the macrophage mannose (Man)-receptor cDNA directs the synthesis of a protein that binds oligosaccharides with either terminal S4GGnM or terminal Man, at independent sites. We now show that the cysteine-rich (Cys-Rich) domain at the N terminus of the Man͞S4GGnM receptor accounts for binding of oligosaccharides with terminal GalNAc-4-SO 4 , whereas calcium-dependent carbohydrate recognition domains (CRDs) account for binding of ligands containing terminal Man. The Cys-Rich domain is thus a previously unrecognized carbohydrate binding motif. Cys-Rich domains have been described on the three other members of the endocytic C-type lectin family of receptors. The structural relationship of these receptors to the Man͞S4GGnM receptor raises the possibility that their Cys-Rich domains also bind carbohydrate moieties and contribute to their function.Glycoproteins such as lutropin (LH) and thyrotropin that bear multiple Asn-linked oligosaccharides terminating with the sequence SO 4 -4-GalNAc␤1,4GlcNAc␤1,2Man␣ (S4GGnM) are rapidly removed from the circulation by an S4GGnM-specific receptor found at the surface of hepatic endothelial cells (1, 2). Precise control of the circulatory half-life of LH is thought to be critical for attaining maximal stimulation of the LH receptor located in the ovary during the preovulatory surge in LH levels. We recently reported (3) that the S4GGnM receptor (S4GGnM-R) isolated from rat liver is closely related to the macrophage mannose receptor (Man-R) isolated from rat lung. However, the liver S4GGnM-R and lung Man-R display marked differences in ligand specificity and binding properties.

Lutropin (LH) and other glycoproteins bearing oligosaccharides with the terminal sequence SO 4 -4-GalNAc␤1,4GlcNAc␤1,4Man-(S4GGnM) are rapidly removed from the circulation by an S4GGnM-specific receptor (S4GGnM-R) expressed at the surface of hepatic endothelial cells. The S4GGnM-R isolated from rat liver is closely related to the macrophage mannose-specific receptor (Man-R) isolated from rat lung both antigenically and structurally. The S4GGnM-R and Man-R isolated from these tissues nonetheless differ in their ability to bind ligands bearing terminal GalNAc-4-SO 4 or Man. In this paper, we have explored the structural relationship between the Man-R and the S4GGnM-R by examining the properties of the recombinant Man-R in the form of a transmembrane protein and a soluble chimeric fusion protein in which the transmembrane and cytosolic domains have been replaced by the Fc region of human IgG1. Like the S4GGnM-R isolated from liver, the chimeric fusion protein is able to bind ligands terminating with GalNAc-4-SO 4 and Man at independent sites. When expressed in CHO cells the recombinant Man-R is able to mediate the uptake of ligands bearing either terminal GalNAc-4-SO 4 or terminal Man. We propose that the Man-R be renamed the Man͞S4GGnM receptor on the basis of its multiple and independent specificities.Glycoproteins bearing Asn-linked oligosaccharides terminating with the sequence SO 4 -4-GalNAc␤1,4GlcNAc␤1,2Man (S4GGnM) are bound by a receptor found at the surface of hepatic endothelial cells that mediates their internalization and transport to lysosomes, where they are degraded (1). The S4GGnM-specific receptor (S4GGnM-R) accounts for the rapid removal of the glycoprotein hormone lutropin (LH) from the circulation and thereby the episodic rise and fall in circulating LH after release from gonadotrophs into the blood (2). The episodic rise and fall in circulating LH levels has been proposed to be essential for attaining maximal biologic activity in vivo (3).We recently described the isolation of a receptor from rat liver that can account for the binding and internalization of LH by hepatic endothelial cells (4). The receptor is closely related to the macrophage mannose receptor (Man-R) (5, 6) on the basis of antigenicity and peptide mapping studies. Yet the S4GGnM-R isolated from liver and the Man-R isolated from lung display markedly different binding properties, suggesting that the two receptors are not identical (4). We have examined this issue by expressing a cDNA, isolated from mouse lung, which encodes the Man-R (6) in Chinese hamster ovary (CHO) cells. We have also examined the properties of a secreted, chimeric fusion protein derived from this cDNA in which the transmembrane and cytosolic domains of the receptor have been replaced by the Fc region of human IgG1 (7). Our results indicate that the Man-R cDNA directs synthesis of a receptor that displays properties characteristic of both the S4GGnM-R and the Man-R when expressed in CHO cells. The properties of the secreted chimeric fusion protein ind...

Certain of the glycoprotein hormones, including bovine lutropin (bLH), bear asparagine-linked oligosaccharides terminating with the sequence So4-4GaiNAcpIJ-4GlcNAcl1-2Mana. To

Differential expression of glycosyltransferases has the potential to generate functionally distinct glycoforms of otherwise identical proteins. We have previously demonstrated the presence of unique oligosaccharides terminating with GalNAc-4-SO4 on the pituitary glycoproteins lutropin (LH), thyroid stimulating hormone (TSH), and pro-opiomelanocortin (POMC). A glycoprotein hormone:GalNAc-transferase and a GalNAc-4-sulfotransferase are present in the pituitary and can account for the synthesis of these unique oligosaccharides on specific glycoproteins. Both transferases are coordinately expressed in a number of tissues in addition to pituitary, including submaxillary gland, lacrimal gland, and kidney, suggesting that additional glycoproteins bearing oligosaccharides terminating with GalNAc-4-SO4 are synthesized in these tissues. In this study we show that while the glycoprotein hormone:GalNAc-transferase and the GalNAc-4-sulfotransferase are coordinately expressed in bovine submaxillary gland, the GalNAc-transferase is expressed in the parotid gland in the absence of the GalNAc-4-sulfotransferase. The relative expression of these two transferases in submaxillary and parotid glands correlates with the presence of unique Asn-linked oligosaccharides on carbonic anhydrase VI (CA VI) synthesized in each of these tissues. The majority of Asn-linked oligosaccharides on CA VI synthesized in submaxillary gland terminate with GalNAc-4-SO4. In contrast, CA VI which is synthesized in bovine parotid gland bears oligosaccharides which terminate predominantly with beta 1,4-linked GalNAc which is not sulfated. The presence of different terminal residues on the Asn-linked oligosaccharides of submaxillary and parotid CA VI thus correlates with the complement of transferases in these glands and suggests differing biological roles for submaxillary and parotid CA VI.

A limited number of glycoproteins including luteinizing hormone and carbonic anhydrase-VI (CA6) bear N-linked oligosaccharides that are modified with ␤1,4-linked N-acetylgalactosamine (GalNAc). The selective addition of GalNAc to these glycoproteins requires that the ␤1,4-N-acetylgalactosaminyltransferase (␤GT) recognize both the oligosaccharide acceptor and a peptide recognition determinant on the substrate glycoprotein. We report here that two recently cloned ␤GTs, ␤GT3 and ␤GT4, that are able to transfer GalNAc to GlcNAc in ␤1,4-linkage display the necessary glycoprotein specificity in vivo. Both ␤GTs transfer GalNAc to N-linked oligosaccharides on the luteinizing hormone ␣ subunit and CA6 but not to those on transferrin (Trf). A single peptide recognition determinant encoded in the carboxyl-terminal 19-amino acid sequence of bovine CA6 mediates transfer of GalNAc to each of its two N-linked oligosaccharides. The addition of this 19-amino acid sequence to the carboxyl terminus of Trf confers full acceptor activity onto Trf for both ␤GT3 and ␤GT4 in vivo. The complete 19-amino acid sequence is required for optimal GalNAc addition in vivo, indicating that the peptide sequence is both necessary and sufficient for recognition by ␤GT3 and ␤GT4.