Structural features of mammalian gonadotropins

becularand

cellular

Endocrinoloey Molecular and Cellular Endocrinology 125 (1996) 3- 19

Structural features of mammalian George

R. Bousfield”,“,

Viktor

Y. Butnev”, R. Russell William T. Mooreb

gonadotropins Gotschall”,

Vanda

L. Baker”,

‘Department of’ Biological Sciences, Wichita State Unioersity, 1845 N. Fairmount, Wichita, KS 67260-0026, USA bProtein Chemistry Laboratory, Medical School qf’ the University of Pennsvlvania, Philadelphia, Penns_vlvania, USA

Abstract There are two species for which both pituitary and placental gonadotropins are readily available, humans and horses. The human gonadotropins are better characterized than equine gonadotropins. Nevertheless, the latter are very interesting because they provide exceptions to some of the general structure-function principles derived from studies on human and other mammalian gonadotropins. For example, separate genes encode the hLHP and hCGB subunits while a single gene encodes eLHP and eCG/3. Thus, eCG and eLH differ only in their oligosaccharide moieties and eLH is the only LH that possesses the O-glycosylated C-terminal extension previously believed to be restricted to chorionic gonadotropins. Truncation experiments involving eLHp and hCG/3 have suggested the C-terminal extension has no effect on receptor binding. However, the largest of three eCG forms which differ only in the extent of O-glycosylation possessed reduced affinity for LH and FSH receptors. This result suggested that effects of O-glycosylation need to be considered when examining the glycosylation differences between eLH and eCG responsible for the lo-fold lower eCG receptor binding affinity compared with that of eLH. Contribution of aAsn 56 N-linked oligosaccharides to the different biological activities of eLH and eCG has been evaluated following selective removal using peptide-N-glycanase digestion of native equine r-subunit preparations. Hormone-specific patterns of glycosylation were observed on ccAsn56 of eLH, eFSH, and eCG. Removal of ctAsn5(’ oligosaccharides increased the rate of subunit association, the extent of association, and receptor binding activity. Some unassociated a-subunit oligosaccharides were identified which may interfere with subunit association because they were more abundant in unassociated subunit oligosaccharide maps than in a total oligosaccharide map. This was most striking in the case of eCGcc in which two minor peaks became the major oligosaccharide peaks detectable in the unassociated eCGcl fraction following association with eLHP and eFSH/3. The biological activities exhibited by hybrid hormones, eLHcl reassociated with oLHB and pLH/3, found to be greater than those of oLH and pLH provided an interesting exception to the general rule that the b-subunit determines the potency of the heterodimer. LH receptor binding activities of eLHP-chimeric ovine/equine cc-subunits suggested that the equine a-subunit N-terminal domain may be responsible for this effect. Equine FSH has higher FSH receptor binding activity than human, ovine, and porcine FSH preparations. This probably results from two factors. First, the presence of the equine a-subunit promotes receptor binding as noted above. Second, the overall - 2 charge of the eFSHP determinant loop, which is less negative that the - 3 observed in other species, results from the presence of an Asn residue at position 88 instead of Asp. This apparently facilitates binding to the FSH receptor. Copyright 0 1996 Elsevier Science Ireland Ltd. Ke_vwor&: Equine; Gonadotropins;

Hybrid hormone: Oligosaccharide;

1. Introduction The classical pituitary glycoprotein hormones, LH, FSH. and TSH, are found in all mammals [1,2]. The fourth member of this family, CG, is produced by the *Corresponding author. Tel.:

+

I 316 9783111; fax: + 1 316

9783772. 0303-7207/96/$15.00 Copyright 8 1996 Elsevier Science Ireland PII SO303-7207(96)03945-7

Receptor

placenta and is found only in primates and equids. In only two species, humans and horses, have all three gonadotropins been well-characterized. Those of the human are better characterized than those of the horse. Nevertheless, eCG has played an important role in elucidating the role of pituitary FSH as it has been frequently substituted for FSH.

Ltd. All rights

reserved

G.R. Bousjield et al. / Molecular and Cellular Endocrinology 125 (1996) 3-19

Fig. 1. Comparison of electrophoretic patterns of human and equine gonadotropins following SDS-PAGE on 15% gels after Coomassie blue staining. The relative mobilities and glycosylation patterns are indicated diagrammatically for each hormone preparation. The G(-and b-subunits are indicated, N-linked oligosaccharides are indicated by a fork, O-linked oligosaccharides are indicated by a straight line. Lane one, 5 pg hLH; lane two, 5 jig eLH; lane three, 5 pg hFSH; lane four, 5 pg eFSH; lane five, 10 pg hCG, lane six, 10 pg eCG.

2. Comparison

of human and equine gonadotropins

Fig. 1 shows SDS-PAGE analysis of equine and human gonadotropin preparations and diagrammatically summarizes the relative sizes of each subunit and their glycosylation status. The a-subunits of most mammalian glycoprotein hormones, including the horse, consist of 96 residues and possess two N-linked oligosaccharides [l]. The human a-subunit gene has a deletion near the amino terminus that results in a 92-residue peptide [3]. During SDS-PAGE the a-subunit band mobility is primarily determined by glycosylation. Thus, the relative electrophoretic mobilities of the equine a-subunits are eLHcr > eFSHcl > eCGa, which correspond to masses determined by matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-MS) of 14254, 14525, and 15435, respectively. The relative mobilities of the human cc-subunits are somewhat different as hCGcc migrates more rapidly than hFSHcc (hLHa > hCGcl > hFSHa). Although most mammalian LH/? -subunits consist of 121 residues, approximately 25 more than their corresponding a-subunits, the former possess only a single N-linked oligosaccharide resulting in a greater electrophoretic mobility. Thus, hLH/? migrates more rapidly than hLHa. The retarded mobility of eLH1 is the result of an additional 28 amino acid residues at.its C-terminus that are 0-glycosylated [4]. Prior to the determination of the eLHP amino acid sequence, it was believed that this C-terminal extension was restricted to the chorionic gonadotropins [4]. However, a single gene encodes both eLHb and eCG/J [5], unlike the primates in which separate genes for LHP and CGB exist [6]. Therefore, evolution of the chorionic gonadotropins in the equidae has taken a different course than that taken in the primate line.

Comparison of the electrophoretic patterns of hFSH and eFSH revealed that eFSH migrated more rapidly than hFSH. Since the polypeptide moieties are very similar, these differences probably reflected smaller oligosaccharides on eFSH than those found on hFSH. The latter has been reported to possess abundant triantennary oligosaccharides [7]. The smaller eFSH oligosaccharides may contribute to its much greater in vitro biological activity in comparison with other mammalian FSH preparations [8,9]. The rapid migrating band in the eFSH lane will be shown to result from the absence of one of the a-subunit N-linked oligosaccharides. The electrophoretic patterns for hCG and eCG resembled each other in that both /?-subunits had slower mobilities than the corresponding a-subunits. However, eCGcc migrated as a ‘ladder’ of 3 to 4 discrete bands while hCGcr migrated as a single band. The hCGP band resembled the hCG@ band except for its slower mobility. The mobility of the eCGP band was even slower than that of eLH/? and it covered an even larger MW range. Lactosamine repeats found in eCG oligosaccharides are major determinants of its electrophoretic pattern.

3. Influence of glycosylation activities

on receptor-binding

of eLH and eCG

3.1. Injkence of lactosamine repeats on the electrophoretic patterns of eCG

We recently characterized three forms of eCG and found that they differed only in the 0-glycosylation of their p-subunits, while possessing virtually identical N-linked oligosaccharides [lo]. Sequential deglycosylation experiments revealed that the eCGcl ladder was

G.R. Bousjield et al. / Molecular and Cellular Endocrinology

A

a subunn (Y+2H)”

(M+H)+’

B p Subunlt (M+H)*’

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125 (1996) 3- 19

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5000

10,000

15,000

20,000

25,000

30,000

35,000

40,000

m/z Fig. 2. Analysis of the effects of endo-p-galactosidase digestion of eLH and three eCG forms by SDS-PAGE and MALDI-MS. Hormone samples were incubated for 24 h in buffer alone or buffer containing 40 mU endo-/?-galactosidase. The samples were split in three and one aliquot serially diluted for radioligand assay, one aliquot applied to a 15% polyacrylamide gel, and the third aliquot desalted using reverse-phase HPLC, vacuum dried, then analyzed by MALDI-MS. (A) MALDI-MS analysis of eLH and eCG preparations as indicated. (B) SDS-PAGE analysis of eLH and eCG forms. Odd numbered lanes consisted of samples incubated in buffer alone. Even numbered lanes consisted of samples digested with endo-a-galactosidase. Lanes one and two, eLH; lanes three and four, eCG-L; lanes five and six, eCG-M: lanes seven and eight, eCG-H: lane nine mol/wt marker proteins as indicated.

determined by the oligosaccharides attached to Asns2, which appear to be more heterogeneous than those on Asn56 [ll]. However, the distinct electrophoretic bands were not detected as individual peaks during analysis by MALDI-MS. Instead, the analysis yielded the same broad peak observed with eLHa, which migrated as a broad band during SDS-PAGE (Fig. 2). A significant difference existed between the electrophoretic patterns and relative mobilities of the hCG and eCG p-subunit bands. The slower migrating hCGP band resembled the faster migrating hCGa band, indicating that O-linked oligosaccharides had little influence on its appearance. The pattern of the eCG/3 band was strikingly different from that of eCGcc in that the former migrated as a very broad band spanning a large &f, range, 40000-68 000 in the case of eCG-M, (the most abundant form), while eCGcr migrated as a series four 22000-25000 M, bands. MALDI-MS analysis confirmed the broad mol/wt range of eCG,&, 2800036000 mass units. Removal of all O-glycosylation sites from eCGP and eLH/? by nicking at LYS~~with endoproteinase Lys-C resulted in a striking change in electrophoretic mobility. The N-terminal eCGB glycopeptide exhibited an electrophoretic pattern consisting of a ladder of three bands similar in appearance to

eCGcl (Fig. 3, compare lanes 2 and 4). The corresponding eLHP N-terminal fragment migrated as three bands that were more closely spaced and exhibited mobilities slightly faster than the eCGP fragment bands. This is consistent with what is known about the Asn” oligosaccharides of these two hormones. The major structures are biantennary, some of the eCGB oligosaccharides possess lactosamine repeats and all terminate exclusively with sialic acid [12-141. The eLHa oligosaccharides are terminated with either sialic acid or sulphate [13,14]. Thus, the electrophoretic patterns and mobilities of eLHP and eCGP appeared to be determined largely by their O-glycosylated C-terminal extensions. A feature of eCG glycosylation is the presence of lactosamine repeats in both its N- and O-linked oligosaccharides [12,13,15]. Digestion of eCG with endo-/?-galactosidase, which cleaves lactosamine repeats, affected the electrophoretic patterns of both eCG subunits, while having no effect on the mobility of eLH subunits, which lack lactosamine repeats (Fig. 2B). The effect on eCGcr was relatively modest, consisting of increased mobility of all its bands and an increased relative abundance of the most rapidly migrating band. The difference in eCGcc mass following endo-p-galac-

G.R. Bousjield et al. / Molecular and Cellular Endocrinology 125 (1996) 3-19

6

tosidase digestion as determined by MALDI-MS was only 290 mass units, about the mass of sialic acid (Fig. 2A). A much more dramatic change in the electrophoretic pattern of eCGa occurred following endop -galactosidase digestion. The eCGj3 bands migrated with increased electrophoretic mobilities and covered a reduced mol/wt range. The electrophoretic pattern of eCGP became more similar to that of hCGP. A less dramatic change occurred in the MALDI-MS analysis. Because of the very broad mass range covered by the eCGj3 signal during MALDI-MS analysis, the values indicated for its mass are only approximate. Of the four eCG preparations that produced a a-subunit signal, only that of endo-P-galactosidase digested eCG-L was visible when all eight analyses were squeezed into a single figure. A 2340-3015 mass unit change resulted from partial deglycosylation of eCG-L and eCG-M (Fig. 2A). There was not enough of either eCG-H preparation to detect this P-subunit. 3.2. Estimation of the number of 0-glycosylation

sites in eLHP and eCG/3 using carbohydrate composition analysis

Although hCGB possesses two N-glycosylation sites compared to a single eCG@ site, the latter exhibited a

97.4 66.2

-

eCGp

eLHp eCGa eLHa eCGp NTP eLHp NTP

Fig, 3. Comparison of the relative electrophoretic mobilities of the N-glycosylated N-terminal fragments of eLH,!I and eCG/3.Samplesof each B-subunit were incubated with 1% endoproteinase Lys-C for 1 h at 37°C then electrophoresed on a 15% polyacrylamide slab gel, then stained with Coomassie blue. Lane one, mol/wt markers as indicated: lane two, eCG; lane three, eLH; lane four, Lys-C-digested eCGp: lane five, Lys-C-digested eLH/I.

Table I Carbohydrate compositions of eCGP and eLH/J’ C-terminal O-glycosylated glycopeptides mol/mol glycopeptide Monosaccharide Galactosamine Glucosamine Galactose Sialic Acid

eCGp 4.7 16.4 24.1 5.2

eLHP 2.4 1.4 3.7 1.6

much slower electrophoretic mobility and covered a much broader mol/wt range than the former. This suggested more extensive 0-glycosylation of eCG/?. A total of eight Ser residues and a single Thr residue are present at the C-terminus extension of hCGP and four of the Ser residues are 0-glycosylation sites [16-181. The eCGP C-terminal extension includes 11 Ser and five Thr residues. Five eCGP 0-glycosylation sites have been predicted from carbohydrate composition data using the amount of GalNAc, since this monosaccharide is found only at the reducing terminus in eCGp O-linked oligosaccharides [15]. This method of estimating the number of 0-glycosylation sites cannot be applied to eLHP, because its N-linked oligosaccharides also possess GalNAc. To eliminate the contribution of the N-linked carbohydrate, we isolated exclusively Oglycosylated glycopeptides from both eLH/? and eCGP by two rounds of Staphylococcus aureus Vg protease digestion and Sephacryl S-200 purification of reduced, carboxymethylated derivatives of these subunits [4]. The monosaccharide composition of the exclusively 0-glycosylated glycopeptides suggested five glycosylation sites in eCGj3 and only three in eLH/? (Table 1) despite the fact that both proteins possess identical amino acid sequences. However, this assumes that all 0-glycosylation sites are 100% occupied with carbohydrate. Because we were able to identify PTH-derivatives of all the Thr and Ser residues of eLHP and eCGb during automated Edman degradation using a Beckman spinning-cup sequencer, this assumption is not true. Hydrophilic amino acid derivatives, such as glycosylated amino acids are not extracted with ethyl acetate. For example, no PTH-Asn and no glycosylated PTHAsn were observed when the N-glycosylation sites of the eCG and eLH subunits were analyzed in the Beckman sequencer indicating 100% glycosylation of these residues. 3.3. IdentiJication of 0-glycosylation sites in eLHj3 and eCGP using solid-phase Edman degradation Since 0-glycosylation sites cannot be predicted from the primary structure of proteins, they must be identified experimentally. Solid-phase protein sequencing has been used to identify glycosylated amino acids [19].

G. R. Bousfield et al. / Molecular and Cellular Endocrinolog~~ 125 (1996) 3- 19

An example of the data obtained by this method is shown in Fig. 4, which illustrates the PTH-amino acid chromatograms of glycosylated Ser residues and non-

B. Glycosylated

A. Non-glycosylated

Residues Ser’ls

I ;er12’

r Ser-CHO

t

I

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Ser ASer

1

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1

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I

+

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hr

,91

I

I

4

8

glycosylated Ser and Thr residues of hCGP. As a result of the Edman degradation chemistry employed by the MilliGen ProSequencer, the most abundant PTHderivatives of Ser and Thr residues recovered were the dehydro forms, which were eluted at 7.62 and 8.07 min, respectively. When these residues were glycosylated, the dehydration reaction was suppressed, resulting in reduced yield of these amino acid derivatives. Glycosylated forms of these amino acid derivatives appeared as new peaks early in the chromatogram that were eluted ahead of the positions of PTH-Ser and PTH-Thr. Two peaks appeared to be associated with glycosylated PTH-Ser, a larger peak indicated with an arrow and a smaller peak eluted immediately after (Fig. 4B). The presence of glycosylated residues in this region of the chromatogram has been confirmed by carbohydrate analysis, however, it was not possible to analyze the individual peaks. When the combination of solid-phase sequencing and carbohydrate composition analysis was applied to eCGb, the PTH-amino acid chromatograms indicated that eight of its 1I Ser residues and four. possibly five, of its five Thr residues were glycosylated (Fig. 5). ThrlJ8 and Ser14” have not been analyzed by this technique because Se?” will not couple to Sequelon arylamine membranes due to steric hindrance accompanying glycosylation of this residue. There appears to be nothing unusual about the protein sequence in this region as a synthetic peptide analog of eCGp residues 121- 149 coupled efficiently to arylamine and was completely sequenced. Based on resistance to carboxypeptidase digestion, Ser14” was earlier reported to be glycosylated [4,20]. The glycosylation status of Thr’4X remains to be determined. Comparison of the O-glycosylation of eLHB with eCGp indicated that the former possessed fewer glycosylated Thr and Ser residues than did eCG//. Only eight to nine sites were identified in eLH/? as compared with 12 or 13 in eCGP. The much greater mol wt of eCG primarily results from larger O-linked oligosaccharide structures attached to more sites than in eLH. The three recently isolated forms of eCG differed by their molecular weights, and were designated eCG-L (low moliwt), eCG-M (medium mol/wt), and eCG-H (high mol/wt), based on their relative sizes [lo]. The differences in their molecular weights resulted only from differences in O-glycosylation. As far as we could tell from a combination of SDS-PAGE, oligosaccharide mapping, and MALDI-MS, the N-linked oligosaccharides attached to each form were identical. Results of experiments involving truncated forms of eLHP and hCG predicted that there would be no effect of the O-glycosylation differences between eLH and eCG and the eCG forms on biological activity [21-231. To our surprise the eCG-H form exhibited significantly lower LH and FSH receptor binding activities (Fig. 6). Truncation of oligosaccharide chains by endo-B-galactosi-

_lLAlJ

ASer

1’2

I

I

8

12

Time (rnii) Fig. 4. Identification of glycosylated PTH-Ser derivatives during analysis of hCGb O-glycosylation sites by automated Edman degradation of an Asp-Pro cleaved hCGj3 derivative that was covalently attached to a Sequelon DITC membrane. (A) PTH-amino acid derivative chromatograms corresponding to the three nonglycosylated Ser residue and single nonglycosylated Thr residue. (B) PTH-amino acid derivative chromatograms corresponding to the four glycosylated PTH-Ser residues. The positions of PTH-Ser, PTH-Thr, PTHdehydro(A)Ser. PTH-dehydro(A)Thr, and glycosylated PTH-Ser (Ser-CHO) are indicated.

7

G.R. Bousjield et al. 1 Molecular and Cellular Endocrinology 125 (1996) 3-19

hCGp

O-glycosylation

eCGP 0-glycosylation

eCGP eLHD O-glycosylation

‘go

1?5

llo

,

1;5

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eLHf3 CGVFRDQPLACAPQASSSSKDP

if57

tap

7

ly5T

lfo

la5

SQPLTSTSTPTPGASRRSSHPLPIKTS

t

CTP-1 I

P-l-D-9

I

c I P-3 Fig. 5. Summary of the O-glycosylation patterns for hCG,4, eCG/?, and eLH/I. The amino acid sequences of the C-terminal extensions of each /I-subunit are shown using the single letter code. Glycosylated residues are indicated by the solid lollipops. Residues with questionable glycosylation status are indicated with a gray lollipop. The only conserved sequence between hCG/I and eLH/CGP is indicated by a box. The arrow points out the position of the last Lys residue. The bars labeled CTP-I, etc. indicate the glycopeptides that were analyzed.

dase digestion significantly increased the activities of eCG preparations, but did not eliminate the significant differences in receptor binding activity between eCG-H and the two other eCG forms and between eLH and all eCG forms. The negative influence of the eCG/? Olinked oligosaccharides was removed when all but one of the O-glycosylation sites were eliminated by cleavage of Asp 120-Pro121by mild acid cleavage. As a result, hybrid hormones consisting of the same cc-subunit preparation associated with either des( 121- 149)eLHP or des( 121- 149)eCGP possessed identical receptor binding activities.

4. Asns6 glycosylation effects on subunit association and receptor binding

It has been reported that chemically deglycosylated FSH and LH subunit preparations associated with one another more rapidly than intact subunit preparations [24]. Deglycosylated gonadotropin preparations possess higher receptor binding activities than the native hormone [25]. The crystal structure of hCG suggests that the Asns6 oligosaccharide could potentially hinder or prevent subunit association in vitro because this oligosaccharide, along with part of the a-subunit hinge loop, must slip through the b-subunit seat belt loop [26,27]. Increased branching of free hCGa oligosaccharides has been reported to be sufficient to prevent it from associating with hCGP [28]. PNGase digestion of

free hCGa removed the impediment to subunit association [29]. In addition, Asn56 oligosaccharide is located close to the putative receptor binding region of hCG, therefore, it can potentially affect receptor binding [26,27]. PNGase selectively deglycosylated native a-subunit preparations at Asn 56 [ll]. This enabled us to investigate (1) the oligosaccharides believed to be the most important in coupling receptor binding to signal transduction; (2) the effects of Asn56 oligosaccharide on subunit association; and (3) the effects of this oligosaccharide on receptor binding. 4.1. Selective deglycosylation of crAsnf6 by PNGase digestion Detection of partial deglycosylation by SDS-PAGE only indicated that it had occurred but could not reveal the site of partial deglycosylation. Site-specific deglycosylation following PNGase digestion has been reported for a-subunit preparations [11,30-321, however, partial deglycosylation at both N-linked sites of hCGP has been reported [33]. Accordingly, we employed solid phase sequencing to examine the status of Asn residues before and after enzymatic deglycosylation. Incubation with CNBr cleaved two conveniently located Met residues found at positions 51 and 77. During automated Edman degradation cycle five, Asn56 was examined and Asng2 at cycle seven. The PTH-chromatograms for native eLHcl indicated glycosylation of both Asn residues by the absence of PTH-Asn accompanied by the presence of several

G.R. Bousjield et al. 1 Molecular and Cellular Endocrinology

125 (1996) 3-19

75

$ a A Ki r ? F .m 0 iE 8 c?

50

25

100

75

&0

50

+-eFSH -+ eLH DG;C;-L + DGeCG-M + eCG-H -)_ DGeCG-H

z

25

0.1

1

10

[Hormone] nM Fig. 6. Effects of glycosylation on the LH and FSH receptor binding activities of eLH and three forms of eCG. (A) LH radioligand assay using [‘251]eCG as tracer and rat testis homogenate as radioligand. (B) FSH radioligand assay using [‘251]eFSH and chicken testis homogenate as receptor preparation. (C) The effects of removal of lactosamine repeats following endo-/J-galactosidase digestion on the FSH receptor binding activities of the same eLH and eCG preparations. The tracer was [‘251]eFSH and the receptor preparation consisted of Chinese hamster ovary (CHO) cells expressing the hFSH receptor.

overlapping peaks that were eluted near the beginning of the chromatogram (Fig. 7). Carbohydrate analysis (not shown) confirmed that these were glycosylated PTH amino acid residues. Following PNGase digestion

of eLHcc, the overlapping peaks disappeared from the cycle five chromatogram and PTH-Asp appeared. This was because PNGase digestion converted Asn to Asp while liberating the oligosaccharide. Reduction and

IO

G.R. Bousfield et al. / Molecular and Cellular Endocrinology

125 (1996) 3-19

Cycle 7

Cycle 5

C. A;P 0.06

0

4

8

0

4

8

Time (min)

G. 1

5

10

15

20

25

30

I

I

I

I

I

I

I

N-Terminal PeptideNHZ-FPDGEFITQDCPECKLRENKYFFKLGVPIYQCKGCCFSRAY~PARSRKTM t CHO Asn56 sf+ 6” 6; 70 7/ Glycopeptide

LVPKNITSFTCCVAKAFIRVTVM CHO 801 85

IV1

Asns2 Glycopeptide

90

i

95

I

GNIKLENH;CQCYCSTCYHHKI-coon

Fig. 7. Characterization of the sites of PNGase cleavage using sequential deglycosylation followed by solid phase sequencing. Pairs of chromatograms show AsnZ6 and As@ of different preparations of eLHr corresponding to automated Edman degradation cycles five and seven. respectively. (A) and (B) native eLHa showing the cluster of peaks indicating glycosylated PTH-Asn. (C) and (D) Asns6-deglycosylated eLHa resulting from PNGase digestion of native eLHcc. (E) and (F) Asns6 and sZ-deglycosylated eLHcc resulting from PNGase digestion of reduced and carboxymethylated partially deglycosylated eLHa. (G) peptides resulting from CNBr-cleavage of eLHG(.

alkylation was required before PNGase removed oligosaccharide from Asn 82. Identical results were obtained with all the remaining equine R-subunit preparations. 4.2. Oligosaccharide mapping Oligosaccharides obtained from PNGase digestion of native equine a-subunit preparations were separated from enzyme and a-subunit by Centricon P-10 ultrafil-

tration. The oligosaccharide mixture was characterized using a high resolution oligosaccharide mapping technique involving high performance anion exchange chromatography along with pulsed amperometric detection [34]. These maps graphically illustrated the heterogeneity of gonadotropin oligosaccharides attached to a single glycosylation site (Fig. 8). The eFSHcl map was typified by five individual major peaks, the eLHa map by a cluster of early eluting peaks, and the eCGcl map by two clusters of peaks.

G.R. Bousjield et al. 1 Molecular and Celhlar Endocrinology

Iy

pI.&GIdAc

y

125 (1996) 3-19

a 2,1+kutuc

J

4

A

eLHcx Asn56 oligosaccharides

-1.o

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-0

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f? n 5 > 2

-0.4

8

-0.2

2 -

-0

eFSHa Asn5” oligosaccharides

-1 .o 0.8 0.6 0.4 0.2

d

,

20

,

/,

/,

I,

40

0

,

80

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Time (zn) Fig. 8. Oligosaccharide maps showing the Asn 56 oligosaccharides obtained by PNGase digestion of native preparations of equine gonadotropin r-subunits followed by ultrafiltration. Ohgosaccharides were separated using a Dionex PA1 column equilibrated with 100 mM NaOH and developed with a gradient of sodium acetate as indicated by the dashed line. The retention times for the commercially available oligosaccharide standards are indicated with arrows. Their structures are indicated at the top of the figure (A) eLHr Asns6 ohgosaccharides; (B) eCG3 Asns6 oligosaccharides; (C) eFSHar Asns6 oligosaccharides.

One of the two major oligosaccharides attached to Asns6 on eCGcl was eluted with the same retention time as a commercially available, biantennary sialylated oligosaccharide that lacked fucose. This corresponded to the major oligosaccharide identified attached to eCGP Asn13 [ 12,131 and a corresponding peak was present in the eCG@ oligosaccharide map. The other major peak is most likely a lactosamine derivative of the first peak since it exhibits the earlier elution time that results when lactosamine repeats are added to

sialylated oligosaccharides [35]. The structures of the other peaks in the chromatogram will require isolation and analysis. The cluster of peaks in the eLHcr AsrP oligosaccharide map were eluted ahead of the neutral biantennary oligosaccharide standards. Carbohydrate composition suggested a monoantennary structure, however, no monoantennary oligosaccharides were commercially available. Since these were the most abundant carbohydrate structures recovered from aAsn56 of oLH [36] and

G.R. Bousjield et al. / Molecular and Cellular Endocrinology

12

125 (1996) 3-19

2.1-

1.4-

0.7-

6 2 tI%

hCGa Asr@ oligosaccharides

0.4-

g $ [I E

0.2-

a

0.6

0 0

20

40

60

80

100

Time (min) Fig. 9. Oligosaccharide maps comparing Asn56 oligosaccharides of eLHlr with those obtained from oLHcc and hCGu. Oligosaccharides were obtained by PNGase digestion of native preparations of these subunits and analyzed as described in Fig. 8. (A) eLHa Asn56 oligosaccharides; (B) hCGa ASII~~oligosaccharides; (C) eCGa As@ oligosaccharides.

orAsn52 of hCG [37], their oligosaccharide maps should be similar to that of eLHcc. Oligosaccharide mapping of oLHa AsrP carbohydrate revealed two groups of peaks in relatively low abundance that were eluted close to the positions of the eLHcr oligosaccharides (Fig. 9C). The hCGa Asn52 oligosaccharide map consisted primarily of peaks with retention times similar to those for eLHa Asns6. The different retention times probably resulted from the fact that hCG oligosaccharides terminate exclusively with sialic acid [12-141 while eLH oligosaccharides terminate with both sialic acid and

sulphate [13,14]. These results provide supporting evidence for monoantennary oligosaccharides attached to eLHol Asns6. Analysis of the major oligosaccharides of eFSH was limited by the absence of purified oligosaccharides that co-eluted with its major oligosaccharides and by the composition analysis which indicated similar carbohydrate structures present at both glycosylation sites whereas the oligosaccharide maps indicated different structures at each site. These will probably prove to be more extensively branched than eLHa oligosaccharides

G.R. Bousjield et al.

Molecular and Cellular Endocrinology

but less extensively branched than hFSH oligosaccharides. 4.3. Reassociation of Asn56 deglycosylated equine cc-subunits

One of the differences between reassociation of LH subunits and reassociation of eFSH subunits is that the latter associate more slowly and to a lesser extent. Whereas, oLH subunits associate efficiently and can be separated from the unassociated fraction with 100% recovery of biological activity [38], at most, 80% eLH activity was recovered following association of eLH c1and p-subunits because unassociated eLHP was also present in the dimer fraction [39]. Reassociation of eFSH subunits required 72 h to reach 50% completion. Accordingly, we compared the abilities of the equine gonadotropin p-subunits to combine with their corresponding native and Asn56-deglycosylated a-subunits. Removal of AsrP oligosaccharides increased the extent of reassociation. Unassociated ASP-deglycosylated (pdg-) eLHcc was almost undetectable (Fig. 1OD). Some 0.5

A. eFSH a+p

E

B. pdg-eFSHa + eFSHP

I

1

C. eLH a+p

I

‘i

125 (1996) 3- 19

13

unassociated pdg-eFSHa was detectable, however there was significantly greater reassociation than that observed with intact eFSHa. There was an improvement in the extent of association with eCGj3, however, some of the pdg-eCGa remained unable to associate with eCGj3. 4.4. FSH receptor-binding activities of reassociated equine gonadotropin subunit preparations

A series of nine hybrid equine gonadotropin preparations were constructed by incubating 80 pug samples of CI- and p-subunits together in 0.5 M Tris-Acetate buffer, pH 7.0 at 37°C for 72 h, conditions that improved the yield of FSH from its subunits. Aliquots of the reassociation mixtures were serially diluted and assayed in several FSH receptor binding assays. Results of binding assays employing chicken testis FSH receptors are shown in Fig. 11. When native subunit preparations were reassociated, two general trends were observed. As expected, hybrids containing eFSH/? were in turn more active than those possessing eLHP, which were more active than those possessing eCG/?. An a-subunit effect was also observable. Within a set of hybrids possessing the same p-subunit, the hybrid possessing eLHcr was more active than the hybrid possessing eFSHa, which was more active than the hybrid possessing eCGcc. The effects of the subunits did overlap so that two eCGP hybrids, eLHcr :eCG/3 and eFSHcl:eCG/?, were more active than the eLHP hybrid, eCGcr:eLH/?. There were two general effects of aAsns6 oligosaccharide removal on receptor binding activity. First, activity increased 3.5- to 5.4-fold for eFSH/I hybrids, except in the case of eLHcr:eFSHp, in which case no change in activity was observed. This increase in biological activity was much larger than the 9 to 28% increase in subunit association that accompanied AsrP deglycosylation. Second, the effect of the cc-subunit was greatly reduced or eliminated. Thus, ccAstP oligosaccharides exerted an inhibitory influence on receptor binding and made a significant contribution to the receptor binding activities of equine gonadotropins.

5. Cbimeric a-subunit studies

0

20

40

0

20

40

Time (min) Fig. 10. Characterization of subunit association using gel filtration on Superdex 75. Equal amounts of each subunit as indicated were dissolved in 0.5 M Tris-acetate buffer, pH 7.0, incubated together for 72 hr at 37°C. Following reassciation, 30 fig samples were chromatographed on a Superdex 75 column that was equilibrated and developed with 0.126 M ammonium bicarbonate at a flow rate of 0.4 mlimin. The two late eluting peaks are acetate and Tris.

The general characteristics of gonadotropins: hormone specificity, species specificity and relative biological activity are determined almost exclusively by the P-subunit [40]. Hybrids created with equine gonadotropin subunits have provided exceptions to some of these rules and may provide interesting models for studying the structural basis for them. For example, when eLH,!I was associated with oLHcr, pLHa, and hCGa, dimerization occurred, however, the dimers were inactive [39]. When the complementary hybrids were

G.R. Bousfield et al. / Molecular and Cellular Endocrinology

125 (1996) 3-19

Corn onent ----- eFSHP

r-7 -eLHP -

25

eCGP

pSubunit

[Hormone] nM Fig. 11. FSH radioligand assay of equine gonadotropin hybrid hormone preparations. The tracer was [ ‘251]eFSH and the receptor preparation was chicken testis homogenate. Hybrid preparations were the same as those analyzed in Fig. 10 serially diluted with radioligand assay buffer. Symbols indicate the cc-subunit preparation and the lines indicate the p-subunit preparation present in each hybrid. (A) Native cc-subunit preparations reassociated with native /I-subunit preparations. (B) Partially deglycosylated a-subunit preparations reassociated with native p subunit preparations.

formed, the oLHa and pLHct hybrids were more active than oLH and pLH, while the hCGa hybrid was less active than hCG. Among the mammalian gonadotropins, the equine and human a-subunits are most divergent in sequence, exhibiting only 77 and 73% sequence identity, respectively, with ovine a-subunit, an

example of a typical mammalian or-subunit [41]. Several equine specific substitutions are found in the C-terminal half of the equine a-subunit and are believed to be responsible for its unusual properties even though an equal number of equine-specific substitutions exist in the N-terminal half. We exploited the fact that Arg-C

G.R. Bou$eld

Endoproteinase kg-c

et al. / Molecular and Cellular Endocrinology

125 (1996) 3- 19

15

s-mc S”“i+OlyS,S

Chromatography w

Fig. 12. Construction of chimeric x-subunits. (A) Separation of N- and C-terminal fragments from o-subunit preparations. (B) Reconstitution of a chimeric a-subunit from fragments obtained from the cl-subunits of two separate species.

protease makes a single nick in the isolated a-subunit long loop, that sulfitolysis reversibly modifies cystine residues, that the N- and C-terminal peptides can be separated by gel filtration, and that the cc-subunit can be reconstituted from its N- and C-terminal fragments [42] to create interspecific chimeric proteins in order to

localize the domain responsible for the activities of equine gonadotropin hybrids (Fig. 12). Fragments were prepared from eLHor and OLHK These were then used to prepare reconstituted oLHcl chimeras, M-subunit equine-ovine and two eLHclNTP:oLHaCTP and oLHaNTP:eLHaCTP, us-

G.R. Bousfied et al. / Molecular and Cellular Endocrinology

16

ing previously reported procedures [42]. The chimeras were reassociated with oLHP, a truncated eLH/3 derivative, des( 121- 149)eLHP, or a truncated hCG/? derivative, des(l12- 145)hCG@, the dimers separated from unassociated subunits by Superdex 75 chromatography and assayed in a rat testis LH RRA using [‘251]eLH as tracer. As shown in Fig. 13, the relative activities of the equine-ovine chimeras were the same regardless of the p-subunit preparation. The eLHaNTP:oLHaCTP hybrids were more active than the oLHaNTP:eLHaCTP hybrids. This suggested that the other equine-specific substitutions should be investigated. The reduced activity of all the hybrids possessing a chimeric a-subunit suggested that recombinant

1 A. oLHP Hybrids lOO-

60-

60-

40-

+

OLH

-A-

oLHdTP:oLHaCTP

6

oLHaNTP:eLHmCTP

+

eLHaNTP:oLHaCTP

20.

B. de-149)eL&

Hybrid;

1 C. des(fl2-145)hdGp Hybrids

60

20

-0-

hCGa + hCGB(-CTP

.,,., 0.1

1

. . ... 10

..,,., 100

. . . ... 1,000

,

,r

l(

0

ng Hormone Fig. 13. LH radioligand assay of oLH, eLH, and hCG derivatives possessing chimeric a-subunits. The tracer was [‘2SI]eLH and the receptor preparation was rat testis homogenate. The activities are compared with those of native a-subunits reassociated with the complementary p-subunit preparation as indicated. (A) Only a single oLH/? hybrid associated with enough efficiency to test in this assay. (B) Hybrids consisting of chimeric subunits associated with truncated eLH/?. (C) Hybrids consisting of chimeric subunits associated with truncated hCG/?.

125 (1996) 3- I9

chimeras might be more useful reagents for the continuation of these studies.

6. eFSH investigations The electrophoretic patterns for hFSH and eFSH resembled each other in that both a- and p-subunits had the same mobilities and overlapped because both subunits possessed two N-linked oligosaccharides (Fig. 1). The FSHa band was stained more intensely than the FSHP, therefore, it governed the appearance of FSH in this analysis. A portion of the eFSH@ population possessed only one of the two potential oligosaccharides. This was indicated by the band migrating ahead of eFSHa. The other eFSH/3 band was obscured by the eFSHcl band. Purified eFSH/3 preparations consist of a single band that co-migrates with eFSHa or a pair of bands. In contrast to the results obtained with eCGcr preparations, MALDI-MS analysis confirmed the presence of two forms in an eFSHP preparation possessing two SDS-PAGE bands. The lower mol/wt component had a mass of 14 322 and the higher mol/wt component a mass of 16223 (Fig. 14A). The mass of an eFSHP preparation having a single, higher molecular weight band was 16 845 (Fig. 14B). Since the electrophoretic mobility of gonadotropin subunits is strongly influenced by the presence or absence of N-linked oligosaccharides, we speculated that the lower mol/wt form of eFSHa might result from the absence of one of its two N-linked oligosaccharides. Analysis of an eFSHP preparation by solid-phase automated Edman degradation revealed the presence of PTH-Asn at cycle five and cycle seven, indicating that Asn’ was partially glycosylated (Fig. 15). The glycosylation site was encountered at two sequencer cycles because of N-terminal heterogeneity that has been observed in all of our eFSHa preparations. Thus, the sequence began at Asn’ and Cys3. Because the latter was disulphide bonded to another Cys residue, no PTH-Cys was observed, giving the false impression of a single amino terminus. At cycle five, the PTH derivative corresponding to the major sequence was Leu, but in the truncated population, Asn’ was analyzed. A pair of overlapping peaks were observed near the beginning of the chromatogram that might be glycosylated PTH-Asn along with PTH-Asn, itself. At cycle seven, the major peak was PTH-Asn despite the high background. In contrast, no PTH-Asn was observed when the N-glycosylation sites of eLHcl were sequenced (Fig. 7A and B). It is not yet known if Asn24 is also partially glycosylated. Partial N-glycosylation is unusual in naturally occurring gonadotropins. The existence of a partially deglycosylated recombinant bFSH/? was reported a number of years ago [43]. When a precious preparation of oFSH used only for iodination in Darrell Ward’s

17

G.R. Eousfield et al. / Molecular and Cellular Endocrinology 125 (1996) 3- f9

A. eFSHp, 2 SDS-PAGE bands (M+2H)+'

(M+H)+' 14,322

100

17000

80

14000

60

10000

z. 40 .Zm

6900 10,000

6,000

5

22,000

18,000

eFSHp, 1 SDS-PAGE band

0.

5

14,000

26,000

30,000

2 c I R

34,000

K

n-h!

8

5

7900

16.645

,100 80

6400

60

g a

4600 10,000

6.000

14,000

16,000

m/z

22,000

26,000

30.000

34,090

Fig. 14. MALDI-MS analysis of two eFSH,Y preparations. (A) An eFSHP preparation that migrated as two bands during SDS-PAGE under reducing conditions. (B) An eFSHB preparation that migrated as a single band.

o o7

Comparison of Mammalian Determinant Loop Amino eFSH@ Cys ” Asn Ser Asp hFSHP Cys” Asp Ser Asp oFSHP Cys”’ Asp Arg Asp bFSHP Cys”’ Asp Ser Asp pFSHP Cys *’ Asp Ser Asp rFSHP Cyss7 Asp Ser Asp

A. Cycle 1

- i 0.06 !/

Asn

0.05 Y

Fig. 16. Comparison of the determinant malian FSHB subunits.

0.04 1

E

I

.

*

-

I

,

1

B. Cycle 5 GILI tz O-O7 cu 0.06 g c

0.05:

x 0.04: Asn-CH> ‘0 2 0.03: a

FSH P-Subunit Acid Sequences Ser Thr Asp Cys” Ser Thr Asp CYS’~ Ser Thr Asp Cys% Ser Thr Asp Cys” Ser Thr Asp CysgJ Ser Thr Asp Cys’)’

Asn 1,1;.;1

I r r t ,I 18 I t 8 o 07jC. Cycle 7

’

I

‘I

8 I,”

0.06<

!...‘... Time (min) Fig. 15. Solid-phase sequence analysis of eFSH/I. The amino acid sequences resulting from N-terminal heterogeneity are shown above the panels with the cycles illustrated in panels A-C boxed. (A) Cycle one, Asn’. (8) Cycle two, Leu5 and Asn’CHO. (C) Asn7CH0 and Thr’.

loop sequences of mam-

laboratory was subjected to SDS-PAGE analysis, its electrophoretic pattern was identical to that of oLH. This caused much consternation because everyone in the laboratory knew that oFSH subunits co-migrated during SDS-PAGE. The p-subunit in that preparation must have possessed only a single N-linked oligosaccharide. Equine FSH has relatively high biological activity compared with other FSH preparations. Combarnous and colleagues [8] found that eFSH was more active than rat, ovine, and porcine FSH in rat FSH receptor binding assays and even more active than these hormone preparations in signal transduction assays. Gordon et al. [9] reported that eFSH possessed greater receptor binding activity than hFSH, oFSH and pFSH preparations using both rat and chicken receptors. There appear to be several reasons that contribute to the elevated activity of this hormone relative to those of other species. First, eFSH possesses the equine a-subunit, that has been shown to confer increased receptor binding activity on oLHP and pLH/I [39]. Second, eFSHcc oligosaccharides appear to be smaller than either hFSHa or pFSHa [9]. Finally, the structure of the determinant loop of eFSH,/3 differs from those of the other FSH preparations. As shown in Fig. 16, there are

18

G.R. Bousjield et al. / Molecular and Cellular Endocrinolog_v 125 (1996) 3-19

only two Asp residues in eFSH/?, the third is replaced by a neutral Asn residue at position 88. Moyle and colleagues [44] have demonstrated that replacement of a positively charged Arg residue at the homologous position 94, with a neutral Gln residue resulted in increased FSH binding by a mutant hCG/FSHp chimera. It is likely that the Asn for Asp substitution in eFSHa contributes to the increased activity of this hormone.

7. Conclusions The equine gonadotropins provide interesting examples of mammalian gonadotropins that exhibit variations in glycosylation that significantly affect their biological activities. These hormones may provide useful reagents for investigating how oligosaccharide modulates gonadotropin action.

Acknowledgements

These studies were supported 29047.

by NIH grant HD-

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[35] Rohrer. J.S. (1995) Separation of asparagine-linked oligosaccharides by high-pH anion-exchange chromatography with pulsed amperometric detection: empirical relationships between oligosaccharide structure on chromatographic retention. Glycobiology 5, 3599360. [36] Weisshaar, G. Hiyama, J. and Renwick, A.G.C. (1990) Site-specific N-glycosyiation of ovine lutropin: structural analysis by one- and two-dimensional IH-NMR spectroscopy. Eur. J. Biochem. 192, 741-751. [37] Weisshadr, G., Hiyama. J. and Renwick. A.G.C. (1991) Site-specific N-glycosylation of human chorionic gonadotropin-structural analysis of glycopeptides by one- and two-dimensional IH NMR spectroscopy. Glycobiology 1, 3933404. [38] Ascoli. M., Liu, W.-K. and Ward, D.N. (1977) Isolation and recombination of ovine lutropin subunits with complete recovery of biological activity. J. Biol. Chem. 252. 5280-5286. [39] Bousfield, G.R., Liu. W.-K. and Ward, D.N. (1985) Hybrids from equine LH: alpha enhances, beta diminishes activity. Mol. Cell. Endocrinol. 40. 69977. [40] Ward, D.N., Bousfield, G.R. and Moore, K.H. (1991) Gonadotropins. In: Reproduction in Domestic Animals (Cupps. P.T., ed.), pp. 25580, Academic Press, San Diego. [4l] Ward, D.N.. Bousfield. G.R., Gordon. W.L. and Sugino, H. (1989) Chemistry of the peptide components of glycoprotein hormones. In: Microheterogeneity of Glycoprotein Hormones (Keel, B.A.. Grotjan, H.E. Jr.. ed. ). pp. l-2 I. CRC Press, Boca Raton. [42] Bousfield, G.R. and Ward, D.N. ( 1994) Evidence for two folding domains in glycoprotein hormone r-subunits. Endocrinology 135, 624-635. [43] Chappel, S.. Beck, A., Nugent, N.. Hyman. L.. Zabrecky, J. and Maurer, R. (1987) Production of bovine follicle stimulating hormone (FSH) by recombinant DNA technology. 69th Annual Meeting of the Endocrine Society. Indianapolis, abstract 130. [44] Moyle, W.R.. Campbell, R.K., Myers, R.V., Bernard, M.P., Han, Y. and Wang, X. (1994) Co-evolution of ligdnd-receptor pairs. Nature 368, 251 -255.