Structure-Function Studies of Human TSH New Advances in Design of Glycoprotein Hormone Analogs Mariusz W. Szkudlinski, Mathis Grossmann, and Bruce D. Weintraub

Recent progress in structure-function studies of glycoprotein hormones has provided new insights into the molecular mechanisms of action of

these hormones and has further supported the concept that physiological

modulation of assembly, bioactivity, and clearance of these hormones is dependent on specific structural components. This review emphasizes

current advances in the structure-function relationships of human TSH, which have contributed to further elucidation of common and hormone

specific features within the glycoprotein hormones family. Novel strategies

are now being applied to investigate the roLe of individual structural elements. The principles discovered in such studies are essential to under-

stand the physiological regulation of hormone bioactivity and allow for the rational design of novel analogs with potential therapeutic applications. 0 1996, Elsevier Science Inc. (Trends Endocrinol Metab 1996;7:277-286).

TSH and the gonadotropins (CG, LH, and FSH) form the family of glycoprotein hor- mones, each composed of a protein and carbohydrate portion. The protein is a het- erodimer of two noncovalently linked pep tide subunits: a common CL subunit and a hormone specific 8 subunit (Pierce and Parsons 1981). The recently clarified crys- tal structure of human chorionic gonado- tropin (hCG) revealed that both the a and p subunits contain a central cysteine knot motif and three peripheral loops (Lapthorn et al. 1994, Wu et al. 1994).

Mariusz W. Szkudlinski and Mathis Gross- mann are at the Molecular and Cellular En- docrinology Branch, National Institute of Di- abetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA; and Bruce D. Weintraub is at the Division of Endocrinology, Department of Medicine, University of Maryland School of Medicine and the Institute of Human Virol- ogy, Medical Biotechnology Center, Balti- more, MD 21201, USA.

Consequently, the glycoprotein hormones are now considered members of the super- family of cysteine-knot growth factors (Ta- ble 1). In contrast to the other growth factors, glycoprotein hormones are all o-B heterodimers stabilized by a unique seg- ment of the f3 subunit described as a “seat- belt”; unlike the other members of the su- perfamily, these hormones are glycosy- lated on both subunits and bind their respective G protein+oupled receptors. The a-8 heterodimer is required for recep- tor binding and biological activity, but the free subunits show minimal activity at the classic receptors for the dimeric hor- mones. The biological effects of free a (Blithe et al. 1991) and hCG8 subunits (Lunar&-Iskandar et al. 1995) however, indicated a possibility of existence of spe- cific receptors for individual subunits. In light of the common (Y subunit and 38% sequence identity between hCG and hu- man TSH (hTSH) B subunit, homology modeling of hTSH was performed and

showed apparent similarities in the global conformation of these two hormones (M.W. Szkudlinski et al. 1996a) (Figure 1). Nevertheless, it should be emphasized that specific features have to be responsi- ble for the unique biological properties of each hormone and remain to be eluci- dated fully in future direct studies of hTSH structure. The carbohydrate com- ponent of glycoprotein hormones consti- tutes 15%35% of their weight and in- cludes two N-linked chains in the (r sub-

unit and one (TSH, LH) or two (FSH, CC) N-linked chains in the p subunit. In addi- tion, the CG 8 subunit has a unique car- boxy-terminal peptide (CTP) with four O- linked carbohydrate chains. This review discusses the biological function of carbo- hydrate chains, protein domains, and in- dividual residues in hTSH. Particular em- phasis is placed on recent advances de- scribing similarities with and differences from other glycoprotein hormones, as well as novel approaches for the design of glycoprotein hormone analogs with clini- cal potential. The citation of the extensive literature on structure-function studies of glycoprotein hormones is not intended to be fully comprehensive, and in a number of instances, the reader is directed to re- cent reviews covering specific aspects in more detail.

* Carbohydrate Structures in TSH Physiology

The carbohydrate chains assume impor- tance in every aspect of the life span of TSH, from early translational events during biosynthesis to its removal from the circulation (Table 2). This is in agree- ment with studies on most other glyco- sylated proteins that showed that func- tions of oligosaccharides are generally multiple and diverse, They are usually related to recognition events between proteins or cells and to modulation of a variety of biological processes, rather than serving a singular function. Overall, the carbohydrates serve comparable functions among the members of the glycoprotein hormone family (Drickam- er 1991, Baenziger 1994). More recent work, however, has shown that the car- bohydrates may have structure-, sub- unit-, and site-dependent roles for hTSH, which are in part different from those for the gonadotropins (Szkudlin- ski et al. 1995a, Grossmann et al. 1995a).

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Table 1. Members of cysteine knot growth factor superfamily

Bioucfive form

I. Clycoprotein hormones CG LH FSH TSH cx Subunit CGS Subunit

II. PDGF family PDGF-AA PDGF-BB PDGF-Al3 PDGF-B/v-sis VEGF

III. Neurotrophin family NGF BDNF NT-3,4

IV. TGF-l3 family TGF-g 1-5 Inhibin A Inhibin B Activin A Activin AB MIS BMP-2,3,4,5,6,8,9,10,12,13 OP-1 NDP DPP-C vg-1 GDF-1

a-CGg Heterodimer CX-LH8 Heterodimer a-FSHl3 Heterodimer (w-TSH8 Heterodimer

I:;

Homodimer Homodimer Heterodimer Homodimer Homodimer

Homodimer Homodimer Homodimer

Homodimer a-PA Heterodimer a-/3B Heterodimer PA Homodimer PA+B Heterodimer Homodimer Homodimers or heterodimers Homodimers or heterodimers Homodimers or heterodimers Homodimers or heterodimers Homodimers or heterodimers Homodimers or heterodimers

BDNF, brain derived growth factor; BMP, bone morphogenic protein; CG, chorionic gonadotropin: DPP, Drosophila decapentaplegic protein; GDF, growth/differentiation factor; MIS, Miillerian inhibitory substance; NDP, Norie disease protein; NGF, nerve growth factor; NT, neurotrophin; OP, osteogenic protein; PDGF, platelet derived growth factor; PDGF-B/v-sis, PDGF-B-like factor; TGF, transforming growth factor: VEGF, vascular endothelial growth factor; and Vg-1 (vegetal cortex protein in Xenopus). Several proteins have alternate names: BMPJ, osteogenin; BMP-6, Vgrl; OP-1, BMP-7; BMP-8, OP-2; BMP-12, GDF-7; and BMP-13, GDF-6.

Biosynthesis

Attachment of N-linked carbohydrates re- quires a specific glycosylation recognition sequence Asn-%-Serf% (Xaa not equal to Pro). Glycosylation starts during trans- lation with the en bloc transfer of a Glc,- Man&GlcNAc),-P-P-Do1 unit to the Asn residue, followed by stepwise processing reactions in the endoplasmatic reticulum and Golgi apparatus, catalyzed by specific

glycosyl transfemses and glycosidases. Consequently, the oligosaccharides’ struc- tures are dependent on the glycosylation machinery of the host cell and the influ- ence of environment, which have impor- tant implications for the production of re- combinant glycoproteins (Goochee and Monica 1990).

Similar to findings for other members of the glycoprotein hormone family, the

Table 2. The role of oligosaccharides in glycoprotein hormones

Assistance in subunit folding and assembly Protection from intracellular degradation Facilitation of secretion from the cell Maintenance of conformation and stability Modulation of biological activity (in vitro and in vivo) Control of differential targeting, clearance and degradation

cotranslational attachment of the oli- gosaccharides is essential for TSH-sub- unit folding and combination, protects the nascent polypeptide from intracellu- lar degradation, and is necessary for the secretion of the mature hormone from the cell (Bielinska and Boime 1995). For example, cx subunit expressed in a bac- terial system lacking the mammalian glycosylation machinery folded poorly and was degraded inside the cell (Tho- takura and Blithe 1995). Incubation of mouse pituitary cells with tunicamycin, an inhibitor of oligosaccharide attach- ment during translation, similarly led to aggregation and intracellular degrada- tion of TSH (Weintraub et al. 1985). Even the selective disruption of single glycosylation sites using site-directed mutagenesis caused significant de- creases of hTSH secretion from tran- siently transfected Chinese hamster ova- rian (CHO) cells (Grossmann et al. 1995a).

Different degrees of terminal processing of the oligosacchatides give rise to a mix- ture of circulating isoforms, which are the main cause of the physiological microhet- erogeneity of these hormones. Accord- ingly, TSH, like other glycoproteins, is pro- duced and secreted not as a single stnrc- ture, but as a set of glycosylation variants or glycoforms (Sergi et al. 1991, Szkudlin- ski et al. 1993). The processed glycopro- tein hormone N-linked oligosaccharides are complex-type structures displaying notable hormone- and species-dependent differences in their terminal residues. Fig- ure 2 shows typical biantennary structures of pituitary bovine TSH (bTSH) termi- nated almost exclusively with SO&Gal- NAc8 l-4GlcNAc8 1, pituitary human TSH (phTSH) oligosaccharides terminated with SO&GalNA@ 1-4GlcNAcp 1, but also with NeuAccu2-3/6Gall31-4GlcNAcl31 and recombinant human TSH (rhTSH) expressed in CHO cells containing only NeuAcaZ-3Gal8 I-4GlcNAc8 1 terminal se- quences. Pituitary hTSH, as well as LH, are unique in that they contain sulfated oligosaccharides, due to the presence of GalNAc-transferase and sulfotransferase in the pituitary thyrotrophs and gonadot- rophs. On the other hand, CG and FSH, like almost all other serum glycoproteins, contain terminal sialic acid residues. Such selective glycosylation may have primarily evolved as a means to control the persis- tence of pulsatile secreted LH and TSH in the circulation and thus avoid receptor

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Figure 1. A ribbon model of human chorionic gonadotropin (hCG) is shown in the left panel and a homology model of hTSH, based on an alignment of hTSHS to a sequence of hCGf3, is shown in the right panel. A molecular model of hTSH was built on a template of an hCG model derived from crystallographic coordinates obtained from the Brookhaven Data Bank (Lapthorn et al. 1994). All coordinate manipulations and energy calculations were done with the use of the CHARh4mlQUANTA package. Subunit coloring: (Y subunit, red; hCGf3, green; and hTSH-f3 blue. For clarity, the carbohydrate chains are not presented. Two parallel peripheral hairpin loops of the a subunit are located in the upper part of these models and two analogous loops of respective p subunits are shown in the lower part. A single u-helical structure in the a subunit (~~40-46) and a characteristic f3 subunit “seat belt” (SW-105) are recognizable in the lower left portion of each model.

desensitization of the target organ (Baen- tributed predominantly to the kidneys, ziger 1994 and 1996). even at the early phase of clearance, in-

Carbohydrate-Dependent Metabolic Clearance

RhTSH produced in CHO cells, which lack the GalNAc-transferase, terminates exclusively in sialic acid (N-acetyl- neuraminic acid, NeuAc) (Figure 2). This appears to be the main reason why the rhTSH with sialylated carbohydrate chains has longer plasma half-life and higher in vivo bioactivity despite lower in vitro bioactivity when compared with the sulfatedlsialylated phTSH or enzy- matically desialylated rhTSH (Szkudlin- ski et al. 1993). Further studies provided

data correlating the carbohydrate com- position with the organ distribution pat- tern of various TSH preparations (Sz- kudlinski et al. 1995b). The rhTSH with highly sialylated oligosaccharides is dis-

dicating that sialylated glycoprotein hor- mones escape from specific receptor- mediated clearance mechanisms in the liver. The early distribution of predomi- nantly sulfated phTSH and bTSH to the liver suggested a role for GalNAc- sulfate-specific receptors in the clear- ance of these hormones, similar to the role of these receptors in the clearance of sulfated bovine and equine LH de- scribed previously by Baenziger (1994 and 1996). Although the properties of receptors clearing asialo- (Gal- or Gal- NAc-terminated) glycoproteins were ex- tensively characterized (Ashwell and Harford 1982), their specific biological roles are still largely unknown. Never- theless, asialo-rhTSH appears to be cleared predominantly by the liver asialo- glycoprotein receptor, as a coinjection of

asialofetuin significantly reduced its liver uptake and increased concentra- tion in the serum (Szkudlinski et al. 1995b). Thus, both sialylated and sul- fated oligosaccharide structures, each to a different extent, determine glycopro- tein hormone distribution as well as plasma levels, and, thereby, the in viva

potency of TSH. Because the sulfated phTSH has a more rapid clearance rate than sialylated rhTSH, it is possible that sulfation contributes to maintenance of highly pulsatile patterns of serum hor- mone concentration after pulsatile re- lease from the pituitary. Accordingly, it has been recently noted that pulsatile administration of TSH might offer supe- rior efficiency with regard to in vivo stimulation of thyroid hormone secre- tion (Leitolf et al. 1995). Thus, both the clearance mechanisms and the pulsatile release of TSH from the pituitary con- tribute to the regulation of thyroid func- tion.

It is now becoming apparent that the effect of glycosylation on protein func- tion cannot be always easily generalized, but, in certain instances, has to be ana- lyzed and understood on an individual basis (Varki 1993). For example, the ter- minal carbohydrate residues at different glycosylation sites may affect metabolic clearance to a different degree. In this respect, the specific oligosaccharide structure of the single chain in the TSH p subunit appears to be more important than the structure of individual carbohy- drate chains of the a subunit (Szkudlin- ski et al. 1995a). Accordingly, Bishop et al. (1995) have observed that the carbo- hydrate residues on the p subunit may play a greater role, compared with those on the a subunit, in determining meta- bolic clearance rate and in vivo potency of FSH, supporting the concept of site- dependent function of carbohydrate chain and their terminal residues. In ad- dition, complex oligosaccharide struc- tures of glycoprotein hormones may be both subunit specific and site specific, suggesting that they are not processed in a completely random fashion; in fact, analysis of the individual N-linked oli- gosaccharides residues of hTSH sub- units has shown different ratios of each carbohydrate structure at each site (Stockell Hartee and Renwick 1992). Consequently, both the oligosaccharide structures and their location in the mol- ecule may influence circulating levels

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v*snv

v t

” A&”

+ Sialic Acid A Galactose l N-Acetylgalactosamine

e Mannose n NAcetylglucosamlne

Figure 2. The sulfated biantennary structure on the left is representative for bTSH; the sulfated and sialylated structure is more typical for pituitary-derived hTSH (the structure in the Middle). Recombinant hTSH expressed in CHO cells is highly sialylated and not sulfated, an example of which is represented by the biantennary structure on the right.

and in vivo bioactivity of the hormone. Such observations further emphasize that the main physiological role of car- bohydrate moieties and their terminal residues lies in the differential targeting and clearance of the hormones. In this respect, it is not surprising that the dis- tribution of hTSH isoforms is under hor- monal control and altered in various dis- turbances of euthyroidism.

Carbohydrate Changes in Various Conditions

Earlier work from our laboratory has shown that the glycosylation pattern of TSH is altered in a variety of physiolog- ical and pathological conditions, result- ing in apparent alterations in the bioac- tivity of the hormone (Gesundheit and Weintraub 1986). TSH from hypothy- roid rats showed a slower metabolic clearance rate, presumably related to its increased sialylation, as demonstrated with [3H]-glucosamine labeling or lectin affinity chromatography analysis. Simi- larly, the distribution of hTSH glyco- forms showed alterations in patients with various deviations from euthyroid- ism. For example, hTSH was more highly sialylated in patients with pri-

mary hypothyroidism, and evidence for hTSH with higher bioactivity was re- ported in patients with resistance to thy- roid hormones (Beck-Peccoz et al. 1985, Beck-Peccoz and Persani 1994). Variable carbohydrate structures of circulating TSH have also been described in TSH- secreting adenomas, the euthyroid sick syndrome, after cranial irradiation, in central hypothyroidism, or after treat- ment with thyrotropin-releasing hor- mone (TRH) (Lee et al. 1995) (Table 3). Even though the specific physiologic roles of such altered glycosylation forms

Table 3. Conditions associated with altered TSH carbohydrate structures

Primary hypothyroidism Central (hypothalamic) hypothyroidism TSH-secreting pituitary adenomas Resistance to thyroid hormone Euthyroid sick syndrome Chronic uremia TRH/octreotide administration Cranial irradiation Intrauterine state Aging

TRH, thyrotropin-releasing hormone.

has not been completely elucidated, it is reasonable to hypothesize that regula- tion of hTSH glycosylation is an adap- tive response contributing to hormone action and pituitary/hypothalamic feed- back. In hypothyroidism, the reactive in- crease in TSH plasma levels would be achieved not only by increased produc- tion and release of the hormone from the pituitary, but the released TSH would have an altered carbohydrate structure that prolongs its plasma half- life. On the molecular level, this may involve a direct control of glycosyltrans- ferases transcription by thyroid hor- mone, as, for example, thyroid hormone status has been shown to modulate 2,6- sialyltransferase mRNA levels in the mouse thyrotroph (Magner 1994). More- over, estrogen appears to modulate ex- pression of glycosyltransferases that synthesize sulfated oligosaccharides of LH (Baenziger 1994). Thus, physiologi- cal control of glycosylation adds another mechanism regulating circulatory con- centrations, including pulsatility, of the hormone.

l Structure-Function Studies of Carbohydrate Chains

Various methods have been used to study the functional role of the oligosac- charides for TSH and the other glyco- protein hormones (Table 4). In general, all these approaches have specific ad- vantages that are balanced by inherent limitations. For example, chemical or enzymatic deglycosylation may be diffi- cult to control and cannot address site- specific roles of the oligosaccharide chains. In contrast, site-directed mu- tagenesis is suited to alter a specific gly- cosylation consensus sequence but could potentially have a carbohydrate- independent effect on the conformation of the hormone. Therefore, these meth- ods should be considered complemen- tary, and only the integration of data obtained with different approaches has recently led to better understanding of the multiple roles of the carbohydrates for hTSH (Table 2). Owing to the limited availability of purified pituitary hTSH, studies on the role of the carbohydrates for TSH have traditionally used pituitary bTSH. The recent availability of rhTSH and the development of new strategies in structure-function studies, as well as further improvement of transient trans-

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Table 4. Methods used to study oligosaccharide function in glycoprotein hormones

I. Prevention of glycosylation during biosynthesis Inhibition of oligosaccharide-precursor attachment (tunicamycin) Expression of subunits in bacterial systems Disruption of glycosylation consensus sequences using site-directed

mutagenesis II. Alteration of glycosylation during biosynthesis

Inhibition of carbohydrate chain processing (swainsonine, castanospermine) Expression in mammalian cells with aberrant glycosylation Coexpression of glycosidases Modification of production conditions Expression in nonmammalian cells (insect cells) Introduction of new glycosylation consensus sequences by site-directed

mutagenesis Generation of chimeric glycoprotein hormone subunits by gene fusion

III. Modification of oligosaccharides of purified hormones Chemical deglycosylation (hydrofluoric acid, trifluoromethane sulfonic acid) Endoglycosidase digestion Sequential exoglycosidase digestion

IV. Dimerization of differently glycosylated subunits prepared by various methods (I-III)

fection systems to enhance expression of hTSH analogs, have further helped to clarify the role of the carbohydrates in the action of hTSH.

An extensive body of literature has been published on the role of the carbo- hydrates on the biological activity of gly- coprotein hormones, producing a wealth of sometimes conflicting results. Part of the reason lies in the limitations of the methods used, but also in the fact that the roles of the oligosaccharides can dis- play some species- or system-dependent variations. Earlier studies on gonadotro- pins and bTSH using chemical and en- zymatic deglycosylation have shown that the oligosaccharides, and predomi- nantly those of the a-subunit, are neces- sary for full in vitro activity of these hormones, but play a much lesser role for high-affinity receptor binding. Par- ticularly, deglycosylated hCG acted as a competitive antagonist in some systems (Bielinska and Boime 1995), whereas partially deglycosylated TSII was shown to posses higher intrinsic activity (Tho- takura et al. 1994). Thus, there is a con- sensus that carbohydrates affect signal transduction at a post receptor-binding step. The precise molecular basis, in the absence of structural information on ligand-receptor complexes, however, re- mains to be elucidated. An indirect mechanism involving a conformational change appears more likely than a direct

interaction of the oligosaccharide with the receptor, as a lectinlike component identified in the hCG receptor is not present in the hTSH receptor. Moreover, receptor bound deglycosylated CG can be converted from an antagonist to an agonist upon antibody binding (Rebois and Liss 1987), further indicating that the absence of glycans in the hormone may lead to abnormal receptor binding in the regions normally protected by oli- gosaccharide chains.

Role of Carbohydrate Residues in Bioactivity

Recent studies have revealed that the effect of terminal sialic acid residues in modulation of receptor activation and signal transduction is different for hTSH compared with hCG and hFSH. We have taken advantage of the oligosaccharide structure of rhTSH, which is different from that of the normal pituitary TSH, to study the relative importance of the sulfated, sialylated, or desialylated car- bohydrate chains in both subunits of TSH (Szkudlinski et al. 1995a). The en- zymatic removal of the terminal sialic acid residues in the cx subunit, but not in the p subunit, increases the in vitro ac- tivity of recombinant hTSH. From this and other studies using sequential enzy- matic deglycosylation (Thotakura et al. 1994), it appears that the role of the terminal sialic acid in the in vitro activ-

ity of hTSH is different compared with the gonadotropins. In the case of gona- dotropins, the removal of negatively charged sialic acid residues may dimin- ish electrostatic repulsion in the process of hormone-receptor interaction (Com- bamous 1992), leading to aberrant re- ceptor binding and ineffective activa- tion. In contrast, receptor binding and signal transduction of desialylated rhTSH occurs undisturbed and even im- proved (Thotakura et al. 1994, Gross- mann et al. 1995a, Fares et al. 1996), suggesting that sialic acid may have function in the proper conformation of hCG and hFSH. The observation that oligosaccharide structures of the (Y sub- unit have a more pronounced role than those of the p subunit in signal transduc- tion, however, is still valid for all glyco- protein hormones (Matzuk et al. 1989, Thotakura et al. 1992, Bielinska and Boime 1995). Combination of site-di- rected mutagenesis with expression in glycosylation mutant cell lines has fur- ther emphasized the unique roles of in- dividual side chains for hTSH activity, different from the gonadotropins (Grossmann et al. 1995a). Previous site- directed mutagenesis on gonadotropin recognition sites revealed that the oli- gosaccharide at (-uAsn52, but not the one at aAsn7’, was necessary for in vitro ac- tivity of hCG and hFSH action (Matzuk et al. 1989, Bielinska and Boime 1995, Bishop et al. 1995). In contrast, the same chain, and specifically its terminal sialic acid residues, markedly attenuated TSH receptor binding and activation (Gross- mann et al. 1995a). As postranslational modifications of carbohydrates regulate glycoprotein hormone activity in normal physiology, modulation of terminal sia- lylation of the aAsn5’ oligosaccharide, which appears more heterogeneous than other side chains, may hence be impor- tant in regulating activity in a hormone- specific manner. Interestingly, an hCG mutant lacking the aAsn52 side chain also had an increased activity at the hTSH receptor, opposite to the effect at its native receptor. This points to differ- ences in receptor-dependent ligand- receptor interactions as a possible expla- nation for these distinct roles, which may have evolved during evolution to maintain the specificity of receptor ligand interactions within the glycopro- tein hormone family. Interestingly, the carbohydrate chain at Asn52 is posi-

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Receptor binding

\

I Bioactiuitg I in uiuo

Finure 3. Relationshins between two structural components of a glycoprotein hormone and its biological characteristics.

tioned at the dimer interface, close to the putative binding site (Lapthom et al. 1994). This fact may help explain why desialylation or removal of this chain affects hormone potency. It is possible that the negatively charged carbohy- drate chain of TSH in this position con- tributes to the interaction with the re- ceptor and may be repelled by the nega- tively charged receptor domain. In this way, negatively charged carbohydrates may limit interaction of the hormone with the receptor in the region contrib- uting to receptor binding and activation. In sum, terminal sialic-acid residues af- fect bioactivity of closely related glyco- protein hormones in a different fashion, dependent on species-, hormone-, and site- specific hormone-receptor interac- tions.

Finally, as described above, the oli- gosaccharides play an important role in tissue distribution and clearance mech- anisms and thus modulate circulating levels and final biopotency in vivo. In general, effects on clearance are far more important for the final in vivo po- tency than on intrinsic activity, and even small changes in clearance can super- sede those observed for the in vitro bio- activity. For example, enzymatically de- sialylated rhTSH has a S- to lo-fold

higher in vitro potency than sialylated rhTSH. Asialo-rhTSH, however, is cleared significantly faster than rhTSH and exhibits very low in vivo activity (Szkudlinski et al. 1993). Similarly, a hTSH mutant lacking the olAsn5* oli- gosaccharide was more active in vitro but was cleared faster and was, there- fore, less potent in vivo than the fully glycosylated hormone (Grossmann et al. 1995a). An important lesson to be drawn from these and similar findings on other glycoprotein hormones is the lack of cor- relation between the effects of carbohy- drates on in vitro and in vivo activities of glycoproteins. Such studies highlight the difficulties of translating results ob- tained by the use of in vitro system into whole organism physiology and illus- trate the importance of determining the activity of such hormones in suitable an- imal models. Moreover, as cited above, the pulsatile pattern of serum hormone concentrations may affect desensitiza- tion and thus may be more important for chronic, compared with acute, hor- mone response. The complex relation- ships between the structural compo- nents of hTSH and biological features of the hormone are illustrated in Figure 3.

l Studies of the Functional Protein Domains of TSH

The precise molecular mechanism of glycoprotein hormone action is still largely unknown, but biological activity requires an intact cu-p heterodimer and initial interaction of several portions of both subunits with the extracellular por- tion of a specific G-protein-coupled re- ceptor leading to the receptor activation and subsequent generation of CAMP as the central second messenger. The hor- mone-receptor interaction appears to be a multistep process involving domains of the N-terminal part of the receptor, which bind the ligand with high affinity; the proposed secondary interaction has low affinity and is responsible for recep- tor activation and signal transduction (Moyle et al. 1994, Ji et al. 1995, Kohn et al. 1995). The search for functional do- mains in the TSH has progressed in par- allel to studies on gonadotropins. It has involved various chemical and biochem- ical modifications and the use of anti- bodies and synthetic peptides to inter- fere with the TSH subunit association or receptor binding (Magner 1990 and 1994). The availability of cloned genes for hTSH p subunit (Wondisford et al. 1988) and human a subunit (Fiddes and Goodman 1981) allowed for expression of bioactive TSH in different cell lines. The initial site-directed mutagenesis study of hTSH (Lash et al. 1992) re- sulted, however, in relatively low levels of mutant analogs insufficient for bio- logical and structural characterization. Recent optimization of hTSH expression and mutagenesis protocols (Grossmann et al. 1995b, Szkudlinski et al. 1995c) have permitted more complete and effi- cient analysis of various functional do- mains of the hormone. Nevertheless, well-known limitations of site-directed mutagenesis relate to possible indirect effects of mutation, for example, by dis- tant conformational effects or aberrant subunit folding and dimerization. Therefore, the interpretation of such data requires caution, confirmation by multiple substitutions, and verification by other complementary methods.

Although multiple glycoprotein-hor- mone analogs have been shown to reduce their expression and receptor-binding abilities, the following spatially related do- mains/residues, which are highly con- served among different species, have been

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u 33-38

Figure 4. Protein domains of hTSH recog- nized as particularly important in hormone function and analog design. A schematic drawing of hTSH is based on Figure 1 (right panel) with subunit coloring: CL subunit, red and hTSH-g, blue. The following highly con- served regions or residues are marked: a-helix (a40-46), a-Lys” (a5’), a-AsnS2 (a5*; glycosy- lation site), a-carhoxyterminus (c&3-92), and the region ~33-38. “Determinant loop” #88- 95) is a part of a characteristic P-subunit “seat belt” @88-105). “Substitution permissive re- gion/residues,” al l-20) and i3-Leu69 (p69) modified to create superagonists of hTSH are located within the opposite loops.

confirmed to be particularly important in receptor binding and activation: a-car- boxy terminus (c&8-92), a-helix (a40-46), “‘determinant loop” in the g subunit (TSHS88-95), and glycosylation site aAsn5* (a52) (Keutmann 1992, Combat- nous 1992, Dias 1992, Ji et al. 1993, Re- ichert 1994, Lapthorn et al. 1994, Moyle et al. 1995) (see Figure 4).The the a-carboxy- terminal residues a88-92 (Yoo et al. 1993) and amino acids a3344 (Leinung et al. 1991, Liu et al. 1993, Xia et al. 1994) have been previously implicated as receptor contact domains of the gonadotropins. Recent studies on hTSH have revealed im- portant differences in the role of these domains for the activity of hTSH com- pared with hCG and hFSH (Grossmann et al. 1995b). Interestingly, a single amino acid, the ultimate aSe?*, was identified to play a unique role for heterodimer expres- sion, receptor binding, and bioactivity of hTSH. Further, coexpxession of mutant a subunits with the l3 subunits of hTSH, hFSH, and hCG showed that specific res-

idues within the second region, a33-44, played a differential role in glycoprotein heterodimer expression. Despite the high degree of structural homology, there are apparent differences in the requirements for heterodimer formation among the gly- coprotein hormones. These differences may have arisen during evolution of these hormones from a common ancestor pro- tein, in parallel to changes in specificity (Moyle et al. 1994).

The identification of functionally im- portant residues and regions occurs, ad- ditionally, by studies of patients with gly- coprotein hormone deficiencies. In con- trast to the TSH g subunit (Hayashizaki et al. 1989, Dacou-Voutetakis et al. 1990, Medeiros-Neto et al. 1996) or the LH S subunit gene (Weiss et al. 1992), point mutations in the common a subunit gene have, to our knowledge, not been described in humans. Such mutations, particularly located at or near cysteine residues, may be expected to result in combined glycoprotein hormone defi- ciencies. Recent findings on the role of residues 33-44 in the a subunit of hTSH (Grossmann et al. 1996) suggest, how- ever, that mutations in these residues could cause a deficiency of a specific glycoprotein hormone. Mutation of indi- vidual residues in the a33-44 region re- vealed that subtle, but significant disso- ciation of postreceptor events, such as receptor binding, CAMP production, and thyrocyte growth, can be achieved with single amino acid substitutions in hTSH. Further experiments will be necessary to assess whether these residues are useful for the development of analogs with se- lectively altered features, for example, with reduced thyroid growth-stimulat- ing activity.

Several other regions and residues have been recently recognized to be im- portant in the modulation of binding af- finity and bioactivity of hTSH and gonad- otropins. For example, the 1 l-20 region in the a subunit with a cluster of basic residues, present in all vertebrates ex- cept hominoids, forms a previously un- recognized domain with the ability to potentiate receptor binding and signal transduction, as well as an important motif in the evolution of glycoprotein hormone bioactivity (Szkudlinski et al. 199% and 1996a). Recent studies on si- multaneous alterations of multiple do- mains revealed that the partial or com- plete loss of interaction with the recep-

tor caused by perturbation in the structure of one domain may be, in cer- tain instances, completely compensated by mutations in an unrelated domain and further supported a concept of the existence of alternative pathways in the multistep process leading to the receptor activation and signal transfer (M.W. Sz- kudlinski et al. 1996b). Moreover, fur- ther studies of various domains employ- ing a combination of alanine with pro- line scanning mutagenesis have revealed the importance of the a-helical confor- mation (a40-46) and the highly con- served aLys 5’ in hTSH bioactivity. The most important functional domains thus far recognized in TSH are depicted in Figure 4. Several areas are analogous to the “composite binding domain of hCG” proposed by Lapthom et al. (1994). Cer- tain of these regions are tightly con- served among different species or ho- mologous hormones, and any modifica- tion of such an area results in analogs with decreased function. In contrast, other not strictly conserved regions, such as human all-20, constitute a group of “modification permissive sites” appropriate to introduce point substitu- tions with no or minimal effect on hor- mone production and immunoreactivity, but with substantial increases in hor- mone binding affinity and bioactivity. Simultaneous alterations in both highly conserved and “modification permissive sites” will provide further insight in the hormone-receptor interaction, revealing cooperative (additive), noncooperative, and mutually exclusive domains (M.W. Szkudlinski et al. 1996b).

. New Strategies in Structure-Function Studies and Design of Therapeutic Analogs

Currently, rhTSH is being clinically tested as a diagnostic and potential ther- apeutic agent in patients with differenti- ated thyroid carcinoma. A recent study demonstrated that the use of rhTSH in humans shows great promise as a safe and effective method to stimulate 13’1 uptake and thyroglobulin secretion without the disadvantages of hypothy- roidism (Meier et al. 1994, Cole et al. 1993). At the same time, several studies have suggested that such rhTSH may not be an optimal stimulator, but that even more effective agonists could be con- structed by genetic and protein engi-

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Seauence I 1 Modification 1

Figure 5. Integration of strategies employed to generate hTSH and gonadotropin analogs.

neering. Therefore, novel superactive analogs of hTSH (both superagonists and long-acting agonists) may be advan- tageous to stimulate t3’1 uptake in the patients. Moreover, a proposed strategy of blocking thyroid receptor-stimulating antibodies in the treatment of Graves’ disease justifies a further search for po- tent human TSH antagonists.

Superactive Analogs

The first superactive analogs of hTSH with major increases in receptor binding affinity, as well as in vitro and in vivo bioactivity (Szkudlinski et al. 1995c), were constructed based on homology compari- son of various sequences, including those we have obtained by sequencing nonhu- man primate glycoprotein hormone genes, available crystallographic data of hCG (Lapthorn et al. 1994), and homology modeling of hTSH. Selective reconstitu- tion of residues present in homologous hormones provided a paradigm for the design of novel therapeutic glycoprotein hormone analogs (Szkudlinski et al. 199% and 1996a). Quadruple mutations in the hTSH with mutated cx subunit (Q13K+E14K+ P16K+Q20K) and addi- tional replacement (L69R) in the hTSH8 subunit showed 95fold higher potency and more than 1.9fold increase in efficacy compared with the in vitro bioactivity of wild-type hormone. Pharmacological properties of such analogs can be opti- mized according to specific therapeutic

requirements, for example, modifying their plasma half-life by expression in cells with different glycosylation capabilities (Figure 5).

Long-acting Analogs

From a therapeutic standpoint, in cer- tain cases, prolongation of half-life of recombinant glycoproteins may be de- sirable, but in other cases, the design of superactive analogs with relatively short half-life may decrease the chance of po- tential receptor desensitization. The ad- dition of carboxy-terminal peptide (CTP) of hCGg containing four sites for 0-glycosylation to the hFSH or hTSH p subunit using gene fusion prolonged plasma half-life and increased in vivo bioactivity of these hormones (Fares et al. 1992, Joshi et al. 1995). Yet other approaches, which are the area of active investigation, include the feasibility of introduction of new glycosylation recog- nition sequences into the subunits. Other possibilities may involve a modi- fication of the glycosylation machinery of the host cell, for example, by cotrans- fection of glycosyltransferases with de- sired properties. An additional approach is based on selective chemical conjuga- tion of the nontoxic amphipatic polymer polyethylene glycol (PEG) to hTSH re- sulting in a significantly lower metabolic clearance rate and retention of the in- trinsic activity (M.W. Szkudlinski, N.R. Thotakura, B.D. Weintraub et al. unpub-

lished). This approach is particularly at- tractive, as PEG may reduce immunoge- nicity of the analog modified by other methods, such as site-directed mutagen- esis. Therefore, further optimization of analogs will likely necessitate a combi- nation of different approaches and de- sign strategies, as illustrated in Figure 5.

Antagonists

For the design of competitive antagonist, the identification of domains separately involved in the receptor binding and acti- vation will be necessary In contrast to the effects of carbohydrate moieties of the hormone, however, there is no experimen- tal evidence for a major dissociation be- tween binding and activation events me- diated by different domains of the protein component. Accordingly, a TSH antago- nist developed by Hoermann et al. (199 1) is a carbohydrate variant of hCG mole- cule, asialoagalacto-hCG. Other strategies for antagonist generation included degly- cosylated hormones (Joshi and Weintraub 1983) and the use of synthetic peptides of TSH subunits (Morris et al. 1988, Has- soun et al. 1995). As relatively high con- centrations (pM> of these antagonists were used to inhibit TSH receptor stimu- lation, further improvement of their effi- cacy will be required. There is, however, currently no universal approach to the de- sign of glycoprotein hormones, and it is apparent that both structure-based de- sign, as well as combinations of various strategies, will be critical for development of novel analogs.

The Future of Glycoprotein Hormone Analogs for Medical Use

The recent findings of antineoplastic (Lunardi-Iskandar et al. 1995) antiviral (Bourinbaiar and Lee-Huang 1995), and other nonclassic effects of glycoprotein hormones and their subunits (Blalock 1989, Blithe et al. 1991) support the pre- diction that new analogs of glycoprotein hormones may be valuable in the future, not only for diagnosis and treatment of thyroid and reproductive disorders, but also in the treatment of other nonendo- crine diseases. In this respect, hTSH re- ceptor mRNA has been found in lym- phocytes and adipocytes, suggesting that these cells are regulated by TSH. Simi- larly, gonadotropin receptor mRNA has been found in nongonadal tissues. Fur- ther insights into the molecular basis of hormone action should help to prepare

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molecules with much higher receptor specificities and optimized clinical effi- cacy. The structural and functional sim- ilarities between domains of glycopro- tein hormones and cysteine-knot growth factors suggest that, despite the differ- ence in the structure of their receptors, these proteins may share common mechanisms of receptor binding and ac- tivation. Successful modifications of gly- coprotein hormones may not be limited to enhancement or antagonism of their classic action, but can be extended by adding novel features; such progress will certainly broaden the analog uses in re- search and therapy. Future design and refinement of glycoprotein hormone an- alogs will be greatly facilitated by devel- opment of novel combinatorial ap- proaches and ultimately will require complete elucidation of the structure of hormone-receptor complexes.

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