glycosylated chicken growth hormone

GENERAL AND COMPARATIVE ENDOCRINOLOGY 68, 408-414 (1987)

Glycosylated Chicken Growth Hormone

Luc R. BERGHMAN,*,' P LENS,* E. DECUYPERE,~,~E. R. K~~HN,$AND F. VANDESANDE*

“Laboratory for Neuroendocrinology and Immunological Biotechnology, Naamsestraat 59, B 3000 Louvain;

fLaboratory for Physiology of Domestic Animals, Kardinaal Mercierlaan 52, B 3030 Louvain; and $Laboratory for Comparative Endocrinology, Naamsestraat 61, B 3000 Louvain, Belgium

Accepted July 17, 1987

In an attempt to raise monoclonal antibodies to chicken pituitary glycoprotein hormones, mice were immunized with the concanavalin A-adsorbed components of a hypophyseal extract. Fusions of these spleen cells with myeloma cells repeatedly yielded hybridoma lines secreting antibodies that recognized specifically the pituitary caudal acidophils, known as the somatotropes. This paper reveals the existence of a glycosylated counterpart of the well-established holoprotein form of chicken growth hormone, similar to what has been established for human growth hormone and prolactin. o 1987 Academic press, IDC.

The electrophoretic heterogeneity of purified GH preparations is a well-established feature (Cheever and Lewis, 1969). The formation of oligomers on the one hand and the occurrence of purification and/or analysis artifacts on the other hand have been -among others-fairly satisfactory expla- nations to date (Lewis et al., 1980; Stolar and Baumann, 1986).

Recently, the discovery of a glycosylated human GH form (G HGH) (Sinha and Lewis, 1986) provided an alternative view on this matter. Very similar modifications of ovine prolactin and human prolactin had been reported earlier (Lewis et al., 1985). Glycosylation of a holoprotein caused an increase of the molecular weight in each of these cases. This led to our search for a pu- tative glycosylated counterpart of the well- known holoprotein form of chicken growth hormone.

MATERIALS AND METHODS Fractionation of the crude hypophyseal extract.

Chicken pituitaries were collected from broiler heads obtained from a processing company and fractionated by a method derived from the protocol of Burke and Papkoff (1980). In brief, pituitaries were homogenized

i Supported by the Belgian Nationaal Fonds voor Wetenschappebjk Onderzoek.

with an Ultraturrax mixer in ice-cold water (20 ml/g tissue). Phenylmethylsulfonyl fluoride (Sigma), solubilized in a small amount of acetone, was added at a 1 mM concentration (Gold, 1967). The pH was adjusted to 9.5 with a saturated calcium hydroxide solution and extraction was performed at 4” for 3 to 4 hr under con- stant magnetic stirring.

After centrifugation, the supematant was brought to pH 4 with metaphosphoric acid and to 0.15 M ammonium sulfate, yielding a first precipitate known to contain GH and prolactin (PRL) (Licht et al., 1977). The remaining supernatant was adjusted to pH 6.5 and 0.8 saturation ammonium sulfate. After overnight stirring, the precipitate, containing the glycoproteins, was harvested by centrifugation.

Con A affinity chromatography. A 30-ml (2.6 X

S-cm) column of concanavalin A (Con A)-Sepharose 4B (Pharmacia) was equilibrated with 20 mM Tris- HCl buffer containing 0.15 M NaCl, 1 mM MnCl,, and 1 mM CaCl, (Ui et al., 1977). The 0.15 M-O.8 saturation ammonium sulfate precipitate was solubilized in the same buffer and desalted by gel permeation chromatography on a PD 10 column (Sephadex G-25 M, Pharmacia) equilibrated with the same buffer and then loaded on the Con A column. After the column was washed to baseline absorption (280 nm) with 5 bed volumes of buffer, the adsorbed glycoproteins were eluted by addition of 0.5 M a-methylglucoside to the starting buffer. The preparation was concentrated by ultrafiltration through an Amicon YM5 membrane.

Preparation of monoclonal antibodies. Six-week- old BALB/C mice were injected four times subcutane- ously with a 50:50 complete Freund’s adjuvant:glycoprotein preparation emulsion, the immunogenic dose being the equivalent of 20 pituitaries. Three weeks after the last subcutaneous injection, an intraperi-

408 0016-6480/87 $1.50 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

GLYCOSYLATED CHICKEN GROWTH HORMONE 409

toneal booster with the same dose of immunogen solubilized in 200 pl of saline was administered.

Three days later, a suspension of spleen cells was prepared and fused with NP3 myeloma cells (Flow Laboratories), by addition of 50% polyethylene glycol solution, according to the protocol provided by GIBCO BRL in the HyBRL prep kit. The fusion mixture was routinely dispersed into four 96-well plates with 3 x 105 thymocytes and hybridoma growth factor (Janssen Pharmaceutics) (Aarden et al., 1985) as the growth-promoting conditions.

Screening was performed immunocytochemically 10 and 17 days after fusion. A home-developed device for the suitable and accurate processing of numerous cul- ture supernatants was used (Berghman and Horsten, 1987).

Among others, the fusate wells containing the GH- like immunoreactive antibodies were expanded, frozen, and cloned by limiting dilution to yield exclusively monoclonal cultures. Ascites fluid was produced in adult male BALB/C mice that were primed with a 0.5-ml pristane (Janssen Pharmaceutics) injection 10 days before the inoculation with 5 x lo6 hybridoma cells in 0.5 ml serum-free medium.

Electrophoresis and immunoblotting. Chemical analysis of the precipitates was performed by sodium dodecyl sulfide (SDS)-polyacrylamide gradient (8-16%) gel electrophoresis (PAGE). Proteins in the gels were transferred to nitrocellulose sheets by semidry electroblotting (Kyhse-Anderson, 1985). Immuno- detection was carried out with rabbit anti-ovine GH antisera (l/1000) as a reference. For the detection of the primary antibody an alkaline phosphatase-conjugated second antibody (Dakopatts, dilution l/400) and nitroblue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate (NBT-BCIP) as chromogenic substrate (Promega) were used (Blake et a/., 1984). Total protein staining was done with Coomassie brilliant blue (Serva, Blau R Nr 35051) and silver staining methods.

To exclude the risk of aspecific adsorption of nonglycosylated components to the Sepharose matrix of the Con A column, an in situ localization of glycoprotein$ on nitrocellulose, based on binding of solubilized lectin, was developed.

Blotting of the glycoprotein-containing gel strips was performed following a routine protocol. The non- occupied protein sites were blocked by a 60-min im- mersion in a 5% bovine serum albumin-Tris-HCl buffer @H 7) solution. After a washing cycle with Con A buffer (Tris-HCl, pH 7, t 1 mM CaCl, + 1 m&4 MI&$) the nitrocellulose strip was incubated in a solution of 4 p.g Con A/ml Con A buffer for 30 mm. Sta- bilization of the glycoprotein-lectin complex-neces- sitated by the strongly alkaline and therefore desta- bilizing immunodetection substrate solution-was achieved by oxidation of the c,arbohydrate moiety of the glycoprotein into aldehyde functions. These active groups were allowed to react with primary amino

functions of the closely associated lectin molecule. The oxidation was carried out with a 10 m?r4 IO,- solution in phosphate buffer (PH 7) for 15 min. The excess active groups after the crosslinking were blocked with a 100 m&4 glycine, pH 7, solution for 1.5 min. Im- munodetection of the bound Con A was carried out with rabbit anti-Con A antiserum (l/5000, Dakopatts), swine anti-rabbit IgG-alkaline phosphatase conjugate (11400, Dakopatts), and NBT-BCIP as substrate system.

Potential glycosylated contaminants of the commer- cial serum albumin source (Sigma fraction V) that was used during the quenching step were batchwise adsorbed to Con A-Sepharose, prior to its use. The presence of such glycosylated albumin derivatives can be ex,plained by nonenzymatic. glycosylation ‘of albumin (Rendell et al., 1986). This purification step has proved to be necessary for background reduction (Glass et al., 1981).

As a control for the method specificity, 1 mg cv- methylglucosideiml was added to the Con A solution. This excess sugar had to be able to block the binding of Con A to solid-phase glycoprotein. If no. aspecific interaction of detection reagents was present, the staining result had to be negative in this instance- which is what actually happened.

One-step isolation of electrophoretically pure glycosylated GH. Because the glycosylated GH components represent only a minor portion,of the total GH population, a quick and high-yield purification had to be designed. Therefore, a monoclonal antibody*based immunoadsorbent to GH was developed after puriflca- tion of the antibodies from high-titer ascitessfluid. Typically 5 mg of monoclonal anti-GH was coupled to CNBr-activated Sepharose 4B. Coupling efficieficy as determined by 280~nm absorption prior to and after coupling usually exceeded 95%.

The final chromatographic purification schedule im- plied the connection of the immunoaffinity column to the outlet of the Con A-Sepharose column during its elution step with ol-methylglucoside. This prevented sample and time loss in manipulation and dialysis of the eluate of the lectin column. Consecutive elutlon of the immunoadsbrbent with a glycine-Hd buffer (pH 2.6) yielded the pure glycosylated forms of OH.

RESULTS

After the alkaline extraction of chicken pituitary tissue, addition of 0.15 M ammonium sulfate and metaphosphoric acid to pH 4 yielded the precipitation of a complex protein mixture (Fig. 1, IB). One of the predominant components of this mixture was identified as the growth hormone band using rabbit anti-ovine GH (NIH) an-

410 BERGHMAN ET AL.

A 0 C A B

FIG. 1. Part I: 816% SDS-polyacrylamide gradient gel, stained for total protein with Coomassie brilliant blue. (A) Low-molecular-weight standard proteins. Ribonuclease A (13,000 Da), chymotryp- sinogen A (25,000 Da), ovalbumin (43,000 Da), and bovine serum albumin (67,000 Da). (B) Ammonium sulfate precipitate (O-O.15 M), solubilized in 10% SDS prior to electrophoresis. The arrow indicates the position of the GH band. (C) Affinity-purified chicken GH. (D) Con A-adsorbed fraction of the 0.15 M-0.8 saturation ammonium sulfate precipitate. The glycosylated GH forms are indicated with arrows. (E) The affinity-purified glycosylated GH forms. During the elution step of the Con A affinity column, the glycosylated GH forms are loaded on the anti-GH affinity column and eluted with a low pH buffer after the routine washing steps. One of the forms has molecular weight that is very close to that of the holoprotein. The larger form has an apparent molecular mass of 25,000 Da. Part II: Immu- noblotting results after semidry blotting of various electrophoretograms. (A) Detection of the holoprotein form in the O-O.15 M ammonium sulfate precipitate with rabbit anti-ovine GH antisera. The use of a mouse anti-chicken GH monoclonal antibody for the detection gives the same result. (B) The glycosylated forms of chicken GH detected among the Con A-adsorbed material with the same antisera. Part III: In situ glycoprotein detection on a blot of purified glycosylated chicken GH. Only the major form is detectable. The minor form might be present in nondetectable amounts or more prob- ablv contains very little sugar residues. This could explain the negligible difference in molecular weight with the protein band of GH.

tisera on nitrocellulose blots of an S-16% SDS-PAGE electrophoretogram (Fig. 1, IIA). The validation of the heterologous antiserum for this purpose was performed on chicken pituitary sections, where the somatotropes are easily recognized as the caudal acidophils (Tixier-Vidal and Follett, 1973; Mikami, 1983). No detectable cross- reactions with other cell types were noticed (Fig. 2). The isolation and identification of this growth hormone-like immunoreactive band as growth hormone-both biologi- cally and biochemically (including partial amino acid sequence determination)- have already been performed in our laboratories and will be reported elsewhere.

Addition of ammonium sulfate to 0.8 saturation and pH adjustment to 6.5 caused

the precipitation of virtually all the protein that was left into solution after the first precipitation step. Purification of this precipitate over Con A-Sepharose reveals that the majority of the second ammonium sulfate precipitate passes unadsorbed through the column, whereas only about 10% was bound to the lectin and eluted afterward by addition of 0.5 M a-methylglucoside (Fig. 3).

SDS-PAGE (S-16%) of this fraction shows three major components. One of these (around 15,000 Da) presumably cor- responds to, the subunits of the gonado- tropins and thyrotropin. The two other bands (22,000 and 25,000 Da) are identified immunologically as GH immunoreactive components (Fig. 1, IIB) using the rabbit


FIG. 2. Immunocytochemical demonstration of the somatotropes in the caudal lobe of the chicken adenohypophysis, using a rabbit anti-ovine GH antiserum in a l/1000 dilution. Goat anti-rabbit IgG (l/20), PAP complex, and diaminobenzidinehydrochloride-H,Oz were used as detection system.

anti-ovine GH antisera on nitrocellulose The presence of GH immunoreactive blots of the electrophoretogram. The larger glycosylated components in the Con A-ad- component of both obviously has a signifi- sorbed fraction was demonstrated again cantly higher molecular weight than the when monoclonal antibodies were raised GH band in the first protein precipitate. using the glycosylated portion of the The glycosylated nature of these compo- second ammonium sulfate precipitate as nents was confirmed by their in situ lectin- antigenic mixture. The resulting hybridoma binding capacity (Fig. I, III).

1 .o

0.2

0

! A230 UFS

0.1

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0.02

10 60

.2 A2a 0”

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lines were divided into four immunocyto-

‘60 300 400 ml-

loading washing eiution

FIG. 3. Elution profne of a routine Con A affinity chromatographic purification of the second ammonium sulfate precipitate. About 90% of the loaded material passes through the column with the void volume. Only a minor portion (around 10%) is adsorbed specifically and eluted by the addition of05 ol-methylglucoside to the starting buffer.

412 BERGHMAN ET AL.

chemical specificity groups. One group of cell lines produced antibodies that recognized a cephalic cell population that was identified as the ACTH cells using a heterologous rabbit anti-human ACTH antiserum as a reference. One other group ret- ognized specifically the castration cells in the chicken pituitary and the third group recognized both castration and thyroidec- tomy cells. But the two different fusion experiments also produced 12 immunocytochemically growth hormone cell-positive hybridoma lines. These antibodies were able to recognize not only the glycosylated GH forms that were part of the antigenic mixture, but also the classic GH band from the first precipitate as is shown by the immunoblotting results. These antibodies have been used to develop an anti-GH immunoadsorbent to allow a simple and effi- cient purification of the proteins we described. It was sufficient indeed to load the glycoprotein fraction on the GH immunoaffinity column as it was desorbed from the lectin affinity column. No interference of the added glucoside with the immunologic binding of the components was noticed. Elution of the immunoaffinity column produced a pure preparation of two glycosylated GH forms (Fig. 1, IE).

DISCUSSION

As expected from previous reports (Leung et al., 1984) most of the chicken GH precipitated at pH 4 and 0.15 M ammonium sulfate, along with a vast variety of other proteins (Fig. 1, IB and IC).

On the other hand, a dozen GH cell- staining cell lines emerged along with some 30 others during the production of monoclonals toward exclusively lectin-bound material. The accuracy of the affinity chromatographic procedure-already mentioned in a number of other reports (Kuhn et al., 1986; Ui et al., 1977; Idler and Hwang, 1978)-was clearly proved for the GH-immunoreactive component of the glycoprotein mixture. Its binding to the

column was the result of specific interaction with the ligand (i.e., the lectin concanavalin A) and had nothing to do with adsorption to the inert matrix (Fig. 1, III). It also predicts the presence of glycosyl and/or mannosyl residues within the carbo- hydrate portion of the described molecule, but more detailed investigation will be necessary to learn more about its exact com- position. The cell lines we mentioned above thus clearly prove the existence of a glycosylated GH cell-specific component within the immunogenic mixture. The physicochemical nature of this component could be-at least partially-elucidated by means of electrophoresis and immunoblotting.

These techniques revealed indeed the presence of two glycosylated minor components on top of the major, faster mi- grating GH band in the holoprotein precipitate. The growth hormone-like nature of these products, suggested by their similar molecular size, was confirmed by their im- munopositive reaction with the rabbit anti- ovine GH antiserum. This antiserum had already been showing an exclusive immunocytochemical specificity for the somato- tropic cell population of the chicken pars distalis.

In addition to this, all the monoclonal antibodies to the glycosylated GH were able to recognize the nonglycosylated counterpart, pleading for strong immunologic re- semblance between both. Moreover, the same immunoadsorbent used in combination with the Con A matrix that was used in these experiments to purify glycosylated growth hormone was used afterward to iso- late a complete growth hormone preparation (i.e., containing glycosylated and nonglycosylated components) from a total pituitary extract. Amino acid sequencing confirmed its predicted identity and revealed no heterogeneity for the amino ter- minus of the molecule (data to be published).

Apart from these immunochemical and


physicochemical similarities, preliminary physiological experiments have also clearly shown that the hormonal activity must be considered at least comparable to that of the nonglycosylated counterpart (data to be published). Pankov and Butnev (1986) reported the same feature for porcine prolactin.

The absence of glycosylated form-specific monoclonals in the fusion results is explained by its very restricted portion of the total amount of GH present in the chicken pituitary. If any antibodies of this specificity were produced, the staining re- sponse in the screening protocol must have been almost negligible, due to an insuffi- cient amount of antigen present.

These results strongly suggest the existence of a glycosylated GH in the chicken pituitary. The similarity to the phenomena described for human GH, human PRL, and ovine PRL is striking (Lewis et al., 1984, 1985; Sinha et al. 1984; Sinha and Lewis, 1986).

Unfortunately, the molecular back- grounds for nonmammalian hormones are not sufficiently known to allow a thorough comparison with, e.g., human GH. The demonstration of a homologous Asn-X-Ser (Thr) sequence, necessary for N-glycosylation (Bahl and Shah, 1977), would greatly elucidate the situation. This might now be- come feasible with the affinity purification protocol described in this paper that allows one to purify a respectable amount of pure material even from a rather poor source.

On the other hand it would be most inter- esting to know whether the glycosylated GH form is not only synthetized in the pituitary but also secreted into the circulation, as is the case for human prolactin (Sinha et al., 1984). The particular importance of this aspect comes from the knowledge that glycosylation of a secretory protein is an im- portant factor for the reduction of its clear- ance from the circulation and thus for the enhancement of its biological activity at the same time (Pankov and Butnev, 1986).

ACKNOWLEDGMENTS

We thank the NIH for the gift of ovine growth hormone and Dr. Balthazart (Liege, Belgium) for the chicken pituitaries we were allowed to use. Mrs. J. Puttemans is gratefully acknowledged for her assis- tance in layout and photography.

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glycosylated chicken growth hormone

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