Evolutionary conservation of the sulfated oligosaccharides on vertebrate glycoprotein hormones that control circulatory half-life.

The circulatory half-life of the mammalian glycoprotein hormone lutropin is controlled by its unique Asn-linked oligosaccharides, which terminate with the sequence SO-4-GalNAcβ1,4GlcNAc. A cluster of basic amino acids essential for recognition of the α subunit by the glycoprotein hormone:N-acetylgalactosaminyltransferase is located within two turns of an α helix (Mengeling, B. J., Manzella, S. M., and Baenziger, J. U.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 502-506). The amino acids within this region are virtually invariant in the α subunits of all vertebrates, indicating that the recognition determinant utilized by the N-acetylgalactosaminyltransferase has been conserved in species ranging from teleost fish to mammals. We demonstrate that the glycoprotein hormone:N-acetylgalactosaminyltransferase and the N-acetylgalactosamine-4-sulfotransferase responsible for the synthesis of these unique sulfated oligosaccharides are expressed in the pituitaries of vertebrates ranging from teleost fish to mammals. Furthermore, we show that Asn-linked oligosaccharides terminating with SO-4-GalNAcβ1,4GlcNAc are present on the α and β subunits of the salmon glycoprotein hormone GTH II. Asn-linked oligosaccharides terminating with SO-4-GalNAcβ1,4GlcNAc are unique structural features of the glycoprotein hormones that have been conserved during vertebrate evolution, suggesting they are critical for the expression of hormone biologic activity.

malian species, whereas oligosaccharides terminating with sialic acid-Gal␤1,4GlcNAc, commonly found on many glycoproteins, predominate on follitropin (2,3). Oligosaccharides bearing terminal ␤1,4-linked GalNAc-4-SO 4 are recognized by a receptor in hepatic endothelial cells, resulting in the rapid removal of glycoproteins bearing these structures from the blood (5)(6)(7). The short circulatory half-life of LH in conjunction with its stimulated release from gonadotrophs by gonadotropin-releasing hormone produces a periodic rise and fall in circulating LH levels, which is thought to be essential for optimal activation of the ovarian LH receptor and fertilization. Thus, in mammals the presence of terminal GalNAc-4-SO 4 is associated with a highly specific function.
The presence of terminal GalNAc-4-SO 4 reflects the activity of a glycoprotein hormone:N-acetylgalactosaminyltransferase and a GalNAc-4-sulfotransferase, which we have shown are expressed in the anterior lobe of the pituitary from a number of mammals (8 -11). In addition to the oligosaccharide acceptor, the N-acetylgalactosaminyltransferase recognizes a protein determinant within the ␣ subunit of the glycoprotein hormones (8,12). All of the information required for recognition of the ␣ subunit by the GalNAc-transferase is contained within a 23amino acid glycopeptide fragment (10). The recognition determinant consists of a cluster of basic amino acids that are contained within two turns of an ␣ helix (13). A number of studies have demonstrated that the region of the ␣ subunit, which includes the N-acetylgalactosaminyltransferase recognition determinant, also contains residues that are critical for combination with the ␤ subunits (14,15) and for binding to and activation of the LH/CG-receptor (16). Thus, a number of distinct functions are dependent on interactions of different proteins with the same region of the ␣ subunit.
Homologues of the glycoprotein hormones have been identified in vertebrates from mammals to teleost fish (17). In mammals, follitropin is responsible for follicular development, LH drives oocyte maturation and ovulation, and thyrotropin regulates thyroid function (18). Similar functions have been attributed to glycoprotein hormone homologues in other vertebrate species. Features of both the common ␣ subunit and the hormone-specific ␤ subunits have been conserved in vertebrate glycoprotein hormones including locations of the Cys and Asn glycosylation sites (17,18). Furthermore, crystallographic studies of human chorionic gonadotropin have revealed that both the ␣ and ␤ subunits have a cysteine-knot motif like that found on nerve growth factor, transforming growth factor-␤, and platelet-derived growth factor (19,20). The residues that are critical for recognition of the ␣ subunit by the N-acetylgalactosaminyltransferase fall within a region of the ␣ subunit that is virtually invariant among vertebrate species. Since the recognition determinant utilized by the mammalian N-acetylgalactosaminyltransferase is present within the ␣ subunits of virtually all vertebrate species, we wished to determine if the glycoprotein hormone:N-acetylgalactosaminyltransferase (8), the GalNAc-4-sulfotransferase (9), and the sulfated oligosaccharide structures, which we have described on LH, thyrotropin, and the free, uncombined ␣ subunits of mammalian species (2,3), are also conserved. The presence of these same sulfated oligosaccharides on glycoprotein hormones of other vertebrates would strongly support the biologic importance of these structures, which have been found to control the circulatory half-life of LH in mammals (6,7).

MATERIALS AND METHODS
Transferase Assays-Pituitaries were dispersed in 5 volumes (vol/wt) of buffer A (25 mM Tris, pH 7.5, 250 mM sucrose, 1 mM EDTA, 0.15% Triton X-100) and sonicated with a Branson sonifier for 2 ϫ 10-s pulses at an output setting of 4 on ice in 1.5-ml microfuge tubes. The homogenates were clarified by sedimentation at 2000 ϫ g for 10 min, and the supernatants were stored at Ϫ80°C until used.
GalNAc-transferase and GalNAc-4-sulfotransferase Product Characterization-[ 3 H]GalNAc was incorporated into the Asn-linked oligosaccharides of hCG by incubating salmon pituitary extracts with 0.4 mM UDP-[6-3 H]GalNAc and agal-hCG under the conditions described above for the GalNAc-transferase assay. The reaction was terminated by the addition of 10% SDS and 1.4 M 2-mercaptoethanol to final concentrations of 0.1% SDS and 50 mM 2-mercaptoethanol and heating at 100°C for 3 min. The reaction was then brought to a final concentration of 50 mM NaPO 4 , 0.4% Nonidet P-40, 1 mM EDTA, pH 8.0, and the oligosaccharides were released by digestion with peptide:N-glycosidase F for 24 h at 37°C. The released oligosaccharides were separated from other reaction products by passage over a Sep-Pak (C 18 ) in H 2 0 and then Sephadex G-15 column (Pharmacia Biotech Inc.). The 3 H-labeled oligosaccharide products were characterized by lectin affinity chromatography on immobilized W. floribunda (11) and digestion with jack bean ␤-hexosaminidase followed by monosaccharide analysis on a single Aminex HPX-87H column (Bio-Rad) eluted with 0.01 N H 2 SO 4 as previously described (1,11).
Isomers of SGGnM-MCO and sulfated monosaccharides were separated on a CarboPac PA1 column (Dionex, 4 ϫ 250 mm) as described (9). The 35 S-labeled SGGnM-MCO assay product was treated with 1.0 N NaOH for 1 h at 80°C to determine stability to alkali (9,24). The sulfated monosaccharide was released from 35 S-labeled SGGnM-MCO assay product using 40 mM HCl at 100°C for 2 h. Sulfated monosaccharides released by mild acid hydrolysis were separated from other degradation products by gel filtration on Sephadex G-10 in 126 mM NH 4 HCO 3 prior to analysis by HPLC as described (9,24).
Characterization of GTH II␤ Asn-linked Oligosaccharides-Asnlinked oligosaccharides were released from 5 mg of GTH II␤ by digestion with peptide:N-glycosidase F and labeled by reduction with B[ 3 H] 4 as described (2). The 3 H oligosaccharides were separated from other reaction products by passage over Dowex AG-50 and Sephadex G-25 in H 2 O. The oligosaccharides were then fractionated by anion exchange HPLC on a MicroPak Ax-5 column (Varian Association) into species that differed in the number and character of their anionic moieties (sulfate and sialic acid) (1,26,27). Fractions containing 3 H oligosaccharides were pooled and desalted by passage over Sephadex G-25 in H 2 O. The oligosaccharides were then characterized by lectin affinity chromatography on immobilized W. floribunda, concanavalin A, and Aleuria aurantia and by ion suppression amine absorption HPLC using either a MicroPak Ax-5 column (Varian) or an Amide 80 column (ToyoSoda) calibrated with authentic standards. Digestions with jack bean, diplococcal, and clostridial glycosidases were performed as described (1)(2)(3)25). Digestion with recombinant GalNAc-4-sulfatase was performed in 50 mM sodium acetate, pH 5.8, 10 mM MnCl 2 , 0.1 mg/ml bovine serum albumin for 48 -72 h at 37°C.

RESULTS
The Glycoprotein Hormone:GalNAc-transferase and GalNAc-4-sulfotransferase Are Expressed in the Pituitaries of All Vertebrates-The sequence of the region of the human ␣ subunit between two sets of double cysteines (Cys 31 -Cys 60 ) is illustrated in Fig. 1. We recently provided evidence (13) that the residues PLR and KK, which are found within two turns of an ␣ helix (19,20), are critical for recognition of the ␣ subunit by the glycoprotein hormone:GalNAc-transferase. Alignment with ␣ subunits from species representing other vertebrate classes ( Fig. 1) reveals that this entire region, including the residues critical for recognition by the glycoprotein hormone:GalNActransferase, are highly conserved. Based on mutagenesis studies (13), substitution of either Ala or Met for the Leu within the Pro-Leu-Arg sequence would not be expected to alter recogni-tion by the GalNAc-transferase significantly. Thus, the ␣ subunits from all vertebrates have the potential to be selectively modified by addition of ␤1,4-linked GalNAc-4-SO 4 to their Asnlinked oligosaccharides. Furthermore, since the number and location of the Asn-linked oligosaccharides on the ␣ subunits have been conserved, the spatial relationship of the oligosaccharides to the recognition determinant should also remain the same on the ␣ subunits from the different vertebrate classes. We therefore examined pituitary extracts from representatives of each class of vertebrate for glycoprotein hormone:GalNActransferase and GalNAc-4-sulfotransferase activities with the same properties as those we have previously described in mammalian pituitary extracts (9 -11).
GalNAc-transferase activity with the appropriate specificity was detected in pituitary extracts from representatives of each vertebrate class (Table I). GalNAc was added to oligosaccharide acceptors on hCG, which contains the recognition motif, but not Trf, which does not contain the recognition motif, at the equimolar concentrations of acceptor protein. Thus, a GalNActransferase, which specifically modifies the oligosaccharides on glycoprotein hormones, is present in extracts from all the pituitaries examined. GalNAc-4-sulfotransferase activity was detected in the same extracts (Table I), indicating that GalNAc added to oligosaccharide acceptors would likely be further modified by sulfate addition.
The specific activities (pmol/mg/h) of the GalNAc-transferase and GalNAc-4-sulfotransferase differed by as much as 6-fold among the extracts from different classes of vertebrates. At least three cell types (gonadotrophs, thyrotrophs, and corticotrophs) express GalNAc-transferase and sulfotransferase in mammalian pituitaries (1-3, 11, 24); furthermore, the levels of both transferase activities in gonadotrophs are modulated by the hormonal state of the animal (28). As a result, differences in specific activity may reflect the proportion of cells expressing GalNAc-transferase and GalNAc-4-sulfotransferase, the levels within specific cell types, hormonal state, and/or other factors. Nonetheless, the levels of both transferases are sufficient to account for the presence of oligosaccharides terminating with GalNAc-4-SO 4 on one or more glycoproteins synthesized within the pituitaries of all vertebrate classes.
Salmon is representative of the lowest vertebrate class in which glycoprotein hormone homologues have been identified. We therefore characterized the GalNAc-transferase and sulfotransferase products produced by salmon pituitary extracts to verify that the salmon transferases synthesize the same structures as their mammalian counterparts.
Agal-hCG and UDP-[ 3 H]GalNAc were incubated with salmon pituitary extract to produce a radiolabeled product for characterization. The 3 H label, which had been incorporated into hCG, was released upon digestion with peptide:N-glycosi-dase F, which releases Asn-linked oligosaccharides intact. The 3 H-labeled oligosaccharides produced by peptide:N-glycosidase F digestion were then isolated by gel filtration. The 3 H-labeled oligosaccharides were bound by immobilized W. floribunda and specifically eluted with 50 mM GalNAc (Fig. 2, panel A), indicating that the oligosaccharide products contained terminal ␤1,4-linked GalNAc (22,23). The linkage and identity of the 3 H-sugar was confirmed by digestion with jack bean ␤-hexosaminidase and analysis on an Aminex HPX-87H column (Fig.  2, panel B). The 3 H-labeled oligosaccharides from a mock digestion eluted with the column void. Digestion with jack bean ␤-hexosaminidase resulted in the disappearance of the 3 Hlabeled material in the void and the appearance of a single peak, which comigrated with GalNAc (Fig. 2, panel B). Thus, the GalNAc-transferase in salmon pituitary transfers GalNAc from UDP-GalNAc to Asn-linked oligosaccharide acceptors in ␤1,4-linkage. Since the salmon GalNAc-transferase displays the same protein specificity as the GalNAc-transferase from mammalian species, adding GalNAc to oligosaccharides on hCG with a much greater catalytic efficiency than to the same oligosaccharides on transferrin (Table I), it most likely utilizes the same recognition motif as the mammalian enzyme.
Asparagine-linked Oligosaccharides Terminating with SO 4 -4-GalNAc␤1,4GlcNAc␤1,2Man␣ Are Present on Salmon GTH II-The studies described above demonstrate that a GalNAc-  transferase and a GalNAc-4-sulfotransferase with the same properties as their mammalian counterparts are expressed in salmon pituitaries. We examined salmon GTH II, a gonadotropin homologue (29), for the presence of Asn-linked oligosaccharides terminating with SO 4 -4-GalNAc␤1,4GlcNAc. Dimeric GTH II and its separated ␣ and ␤ subunits were analyzed using a monoclonal antibody specific for the sequence SO 4 -4-GalNAc␤1,4GlcNAc␤1,2Man␣ (anti-S4GGnM) following electrophoresis and transfer to Immobilon-P (Fig. 4, panel A). Anti-S4GGnM did not react with hCG, which bears oligosaccharides terminating with sialic acid-Gal (lane 1) but reacted strongly with the ␣ and ␤ subunits of bovine LH, both of which bear oligosaccharides terminating with GalNAc-4-SO 4 (1) (lane 2). Intact GTH II containing both ␣ and ␤ subunits also reacted with anti-S4GGnM (lane 3). Since the ␣ and ␤ subunits of intact GTH II were not resolved, it was not possible to determine if one or both subunits contained oligosaccharides reactive with anti-S4GGnM. However, when GTH II ␣ and ␤ subunits, which had been previously separated by reverse phase HPLC (29), were examined, both were reactive with anti-S4GGnM (lanes 4 and 5, respectively) The GalNAc-4-sulfotransferase is highly specific for the terminal sequence GalNAc␤1,4GlcNAc␤, and incorporation of [ 35 S]SO 4 into oligosaccharides by partially purified GalNAc-4sulfotransferase can be used to identify glycoproteins bearing oligosaccharides that terminate with this structure. GalNAc-4sulfotransferase purified from bovine submaxillary glands was used for this purpose (Fig. 4, panel B). Sulfate was not incorporated into hCG bearing Asn-linked oligosaccharides with terminal ␤1,4-linked Gal (lane 2), whereas sulfate was transferred to hCG bearing Asn-linked oligosaccharides with terminal ␤1,4-linked GalNAc (lane 3), demonstrating the specificity of the sulfotransferase preparation. subunits of GTH II bear terminal ␤1,4-linked GalNAc-4-SO 4 and/or ␤1,4-linked GalNAc. These studies did not indicate what proportion of these oligosaccharides have this modification or their underlying structure.
A Major Fraction of the Asn-linked Oligosaccharides Present on Salmon GTH II␤ Terminates with ␤1,4-linked GalNAc or GalNAc-4-SO 4 -Studies were undertaken to establish both the distribution and structural features of the Asn-linked oligosaccharides on GTH II␤. This would allow us to determine if these structures are closely related to the sulfated oligosaccharides present on LH and thyrotropin from mammals and if they are major components on at least one of the GTH II subunits. Oligosaccharides were released by digestion with peptide:Nglycosidase F and radiolabeled by reduction with KB[ 3 H] 4 . Fractionation by anion exchange HPLC on a MicroPak Ax-5 column yielded three predominant species designated N0, N1, and S1 which constituted 20, 27, and 29%, respectively, of the radiolabeled species obtained from GTH II (Fig. 5). Characterization of the oligosaccharides in the three fractions by a combination of lectin affinity chromatography, ion suppressionamine adsorption HPLC, and digestion with glycosidases as previously described (1,25) established that the structures in Table II are present on GTH II␤ in the proportions indicated. Each of the oligosaccharide fractions was quantitatively bound by A. aurantia lectin, indicating the presence of an ␣1,6-linked fucose on the Asn-linked GlcNAc (30). Digestion with New Castle Disease Virus neuraminidase, which is linkage-specific (31), indicated that each of the oligosaccharides in the N1 fraction contained a single ␣2,3-linked sialic acid. The sialic acid was linked exclusively to Gal since no increase in binding to immobilized W. floribunda was seen following neuraminidase digestion. The anionic moiety present on the oligosaccharides in the S1 fraction was confirmed to be a 4-linked sulfate by digestion with human GalNAc-4-sulfatase (32,33), which converted these oligosaccharides to neutral species. Prior to digestion with GalNAc-4-sulfatase, 6% of the oligosaccharides in the S1 fraction were bound by immobilized W. floribunda, whereas following digestion Ͼ77% of the oligosaccharides were bound by W. floribunda, indicating that removal of the sulfate exposed a ␤1,4-linked GalNAc. The linkage of the terminal GalNAc was confirmed by comparing the effects of jack bean and diplococcal ␤-hexosaminidase digestion of the neutral oligosaccharides produced by sulfatase digestion of S1. Digestion with jack bean ␤-hexosaminidase abolished binding by immobilized W. floribunda, whereas digestion with diplococcal ␤-hexosaminidase did not affect binding. We have previously shown that jack bean but not diplococcal ␤-hexosaminidase will release ␤1,4-linked GalNAc (1). The digestions were also monitored by ion suppression-amine adsorption HPLC, which demonstrated that the oligosaccharide products comigrated with the appropriate standards prepared from mammalian glycoprotein hormones (34,35). Based on the specificities of the glycosidases and comigration of digestion products with authentic standards during ion suppression-amine adsorption HPLC, the major oligosaccharides present in the N0, N1, and S1 fractions were assigned the structures shown in Table II. The proportion of the total oligosaccharide population obtained from GTH II␤ is shown for each oligosaccharide species. The oligosaccharides shown in Table II account for 75% of the oligosaccharides present on GTH II␤. The oligosaccharides bearing GalNAc and/or GalNAc-4-SO 4 are identical to those previously described on mammalian glycoprotein hormones (1-4) and represent a major fraction of the oligosaccharides present on GTH II␤. DISCUSSION Homologues of the glycoprotein hormones have been described in representatives of each class of vertebrate (17). The glycoprotein hormones are dimeric proteins consisting in each case of a common ␣ subunit and a hormone-specific ␤ subunit. Fish are the only known example in which two forms of ␣ subunit differing in amino acid sequence are present (17). Recent crystallographic studies have revealed that both the ␣ and ␤ subunits of the glycoprotein hormones have cysteine knot motifs, placing them in a family of proteins that includes nerve growth factor, transforming growth factor-␤, and platelet-de- FIG. 5. Anion exchange HPLC profile of Asn-linked oligosaccharides from salmon GTH II␤ subunit. Asn-linked oligosaccharides from salmon GTH II␤ subunit were released by treatment with peptide:N-glycosidase F and radiolabeled at their reducing termini with [ 3 H]borohydride as described under "Materials and Methods." 3 H-Labeled oligosaccharides were subjected to anion exchange HPLC on an AX-5 column. Shown are 3 H-labeled oligosaccharide elution positions corresponding to authentic standards bearing no charged residues (N0), one (N1), or two (N2) sialic acid residues; one (S1), two (S2), or three (S3) sulfate residues; one sulfate and one sialic acid residue (SN), one sulfate and two sialic acid residues (S1N2), or two sulfates and one sialic acid (S2N1). rived growth factor (19,20). A number of structural features have been highly conserved among the glycoprotein hormone homologues including 1) the cysteines that form five and six disulfide bonds in ␣ and ␤ subunits, respectively, 2) the location and number of Asn-glycosylation sites, and 3) individual amino acids that are thought to be involved in dimer formation, receptor binding, and receptor activation.
The current studies demonstrate that yet another structural feature of the glycoprotein hormones is highly conserved through evolution from fish to mammals: the presence of Asnlinked oligosaccharides terminating with the sequence SO 4 -4-GalNAc␤1,4GlcNAc␤1,2Man␣. We have determined that Gal-NAc-transferase and GalNAc-4-sulfotransferase activities with the same specificities and properties as the mammalian enzymes are expressed in representatives of each class of vertebrate. In addition, we have shown that salmon GTH II bears Asn-linked oligosaccharides with this terminal sequence. Taken together with the fact that the amino acids that we have shown are critical for recognition of the ␣ subunit by the glycoprotein hormone:GalNAc-transferase are conserved in the ␣ subunits of each class of vertebrate, it is highly likely that individual members of the glycoprotein hormone family in each class of vertebrate bear Asn-linked oligosaccharides terminating with SO 4 -4-GalNAc␤1,4GlcNAc␤1,2Man␣.
The central region of the ␣ subunit, a stretch of 30 amino acids between two sets of double cysteines (see Fig. 1) and the carboxyl-terminal region of the ␣ subunit are highly conserved through evolution from fish to mammals (17). Various residues within the central region have been shown to be important for interaction with and activation of glycoprotein hormone receptors such as the LH receptor (16) and for dimer formation (14,15). The remarkable degree of sequence conservation in this region may thus reflect the requirement to mediate a number of interactions with different ␤ subunits and receptors. The requirement for recognition of specific basic residues within this region by the glycoprotein hormone:GalNAc-transferase adds yet another restriction to the changes in amino acid sequence, which would be tolerated in this region.
We have previously shown that the presence of oligosaccharides terminating with SO 4 -4-GalNAc␤1,4GlcNAc␤1,2Man␣ on LH plays an important role in mammals by reducing the circulatory half-life of LH in the blood (6). LH bearing oligosaccharides terminating with SO 4 -4-GalNAc␤1,4GlcNAc ␤1,2Man␣ is bound by a SO 4 -4-GalNAc-specific receptor, which is expressed on hepatic endothelial cells and removed from the circulation (5). This rapid removal is essential to produce the pulsatile rise and fall characteristic of circulating LH and is thought to be critical for maximal receptor activation. The presence of the sulfated oligosaccharides on glycoprotein hormones from all vertebrate classes suggests that these structures play a similar and critical biologic role in all vertebrate classes. Notably, Fontaine et al. (36) observed that the metabolic clearance rate for hCG, which bears oligosaccharides terminating with sialic acid-Gal, was 20 times slower than for carp GTH. If carp GTH, like salmon GTH, bears oligosaccharides terminating with SO 4 -4-GalNAc␤1,4GlcNAc␤1,2Man␣, this rapid rate of clearance could reflect the presence of a SO 4 -4-GalNAc-specific receptor in the liver of fish similar to that found in mammals.
The presence of terminal SO 4 -4-GalNAc␤1,4GlcNAc␤1,2-Man␣ is characteristic of vertebrate glycoprotein hormones in species ranging from fish to mammals. Thus, expression of glycoprotein hormone biologic activity must at some juncture require the presence of oligosaccharides terminating with SO 4 -4-GalNAc␤1,4GlcNAc␤1,2Man␣. Our previous studies indicate the most likely role of these structures is to regulate the circulatory half-life of individual glycoprotein hormones through the GalNAc-4-SO 4 receptor; however, other equally critical functions are also possible. In addition, the presence of these same structures on other glycoproteins such as carbonic anhydrase synthesized in salivary glands (25) and proopiomelanocortin (24) raises the possibility that these structures will serve other roles on other glycoproteins.