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Endocrinology Vol. 139, No. 2 527-533
Copyright © 1998 by The Endocrine Society

ARTICLES

Characterization of Human Lutropin Carboxyl- Terminus Isoforms1

Jacques Pantel, Philippe Robert, Frederic Troalen, Michelle Kujas, Dominique Bellet and Jean-Michel Bidart

Département de Biologie Clinique, Institut Gustave-Roussy (J.P., P.R., F.T., D.B., J.-M.B.), 94805 Villejuif, France; Laboratoire d’Immunologie CNRS URA 1484, Faculté des Sciences Pharmaceutiques et Biologiques (D.B., J.-M.B.), 75006 Paris, France; and Service d’Histologie-Embryologie-Cytogénétique, CNRS URA2115, CHU Pitié-Salpétrière (M.K.), 75013 Paris, France

Address all correspondence and requests for reprints to: Pr. J. M. Bidart, Département de Biologie Clinique, Institut Gustave-Roussy, rue Camille Desmoulins, 94805 Villejuif, France. E-mail: bidart{at}igr.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human lutropin (hLH) exhibits both carbohydrate and peptidic heterogeneities that affect its biological potency and the duration of its activity in vivo. Peptidic changes within the hLH ß-subunit are characterized as intrachain proteolytic nicking and carboxyl terminus heterogeneity. To date, the carboxyl terminus of hLHß appears to end at either position Gln114 or Gly117, as determined by sequencing of purified subunit. Furthermore, the complementary DNA for hLHß predicts a protein containing an additional peptidic stretch, which would make the ß-subunit 121 residues long. This extension may be responsible for the particular intracellular behavior of hLHß. To investigate the carboxyl terminus polymorphism of natural hLHß, monoclonal antipeptide antibodies were raised against a synthetic peptide mimicking the 104–119 portion of hLHß. One antibody, designated LHP09, was found to specifically react with the recombinant hLHß ending at position hLHß[Leu119] but not with other recombinant forms ending at [Ser116], [Phe120] or [Leu121]. Immunochemical analysis of hLH, either pituitary or urinary in origin, indicated that only pituitary hLH contains a Leu119-ending form of hLHß. Finally, immunohistochemical detection was performed using LHP09 and showed specific staining of a normal adult pituitary gland. These observations support the in vivo existence of intrapituitary molecular forms of hLHß ending at various positions between Gln114 and Leu121.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HUMAN LUTROPIN (hLH) in the pituitary gland is composed of an {alpha}-subunit identical to that of the other glycoprotein hormones, including human CG (hCG), hFSH, and hTSH, and of a ß-subunit that confers its hormonal specificity (1). Lutropin plays an important role in the development of the growing follicle and in the maturation of the oocyte (2). Purified as well as pituitary hLH exhibit considerable molecular heterogeneity affecting both the biological potency and the duration of activity of the hormone in vivo (3). Although much of the heterogeneity of hLH is associated with its carbohydrate moiety, peptidic changes were characterized as allelic variation at residues ßTrp8 and ßIle15 (4), intrachain proteolytic nicking (5, 6), and carboxyl-terminal peptide heterogeneity (7). Indeed, to date there is no agreement between laboratories regarding the carboxyl terminus of hLHß that appears to end at either position Gln114 (8) or Gly117 as determined by conventional protein sequencing methods (9, 10). Furthermore, the complementary DNA for hLHß predicts a protein containing four to six addition-al carboxyl terminal amino acids, which would make the ß-subunit 121 residues long (11). This additional peptidic stretch appears to play a critical role in posttranslational processing of the ß-subunit of hLH and its efficiency when combining with the {alpha}-subunit (12).

Antibodies to synthetic peptides mimicking predetermined amino acid sequences of a protein have proven useful for both understanding the structure of proteins and analyzing the molecular forms of the hormones. Antibodies directed against synthetic peptides mimicking different regions of hLHß and hFSHß were used to analyze the structure of these hormones (13, 14). A series of site-specific monoclonal antibodies (mAbs) directed against synthetic peptides analogous to carboxyl terminus of either the {alpha}-subunit or the ß-subunit of hCG, hLH, and hTSH have been used to map topographic antigenic domains and to analyze the various molecular forms of these hormones (15, 16).

In an attempt to investigate the carboxyl terminus molecular heterogeneity of the ß-subunit of hLH, we raised monoclonal antibodies against a synthetic peptide mimicking the 104–119 region of hLHß. One antibody was found to specifically react with the native form of a recombinant hLHß with a carboxyl-terminal ending at position Leu119. Immunochemical analysis of a pituitary preparation of hLH and immunohistochemical staining of pituitary tissue indicated that the Leu119-ending form of hLHß is present at the pituitary level. In contrast, the presence of this particular form was not detected in hLH prepared from the urine of postmenopausal women, indicating that it does not circulate and/or it is degraded in the kidney. These results provide new insights into the metabolism of hLH and are of interest for the analysis of recombinant gonadotropins used in human reproduction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Purified hLHß (AFP-3282B) and hLH (AFP-8270B) were kind gifts from the National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health (Bethesda, MD). Preparations of recombinant hLHß with a carboxyl terminus ending at position Ser116 (hLHß[Ser116]), Leu119 (hLHß[Leu119]), Phe120 (hLHß[Phe120]), and Leu121 (hLHß[Leu121]) were derived from the purification by RP-HPLC of recombinant hLH, and they were kindly provided by IRCS-Serono (Rome, Italy). Na125Iodine was obtained from the Commissariat à l’Energie Atomique (Saclay, France). The Iodogen method was used to label ß-subunits and monoclonal antibodies to a specific activity of 50 µCi/µg (1 Ci = 3.7 x 1010 Bq) and 20 µCi/µg, respectively. Selection and characterization of monoclonal antipeptide antibodies, designated LHP03 and LHP04 and recognizing the hLHß(43–52) and hLHß(110–117) portions respectively, were previously reported (17). Monoclonal antibody 518B7, generated against bovine LH, reacts with an epitope present on the LHß/CGß of most species and was kindly provided by Dr. J. F. Roser (18).

Peptide synthesis
Four peptides encompassing the hLHß (104–116), (104–119), (104–120), and (104–121) carboxyl terminus were synthesized by a conventional solid-phase method using an Applied Biosystems (Foster City, CA) model 431A peptide synthesizer (13). After completion of synthesis, the identity and purity of each peptide were checked by 1) amino acid analysis on an {alpha}-LKB analyzer; 2) HPLC; and 3) microsequence analysis of each HPLC peak on an automated Applied Biosystems 477A protein sequencer. The hLHß (104–119) peptide was conjugated to keyhole limpet hemocyanin using carbodiimide as the coupling agent. A synthetic peptide encompassing the hLHß (43–52)-(110–117) portions was previously described (13). The amino acid sequences of the different peptides are presented in Table 1Go.


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  		Table 1. Carboxyl-terminal amino acid sequences of hLHß isoforms and peptides used in this study

 
Production and selection of antipeptide antibodies
Six-week old BALB/c mice were immunized with the hLHß (104–119) peptide-carrier conjugate (18). During the immunization procedure, the sera of mice were tested at various dilutions for the presence of antipeptide antibodies by enzyme-linked immunosorbent assay (ELISA) with the hLHß (104–119) unconjugated peptide coated onto polystyrene wells. Three days after an iv boost, spleen cells of animals displaying serum antipeptide antibodies were fused with NS1 myeloma cells using 40% polyethylene glycol (Mr 1,000). Hybridoma colonies that produced mAbs specifically reacting with the 125I-labeled recombinant hLHß ending at position Leu119 (125I-hLHß[Leu119]) in an RIA were selected and subsequently cloned. Binding experiments were performed in 50 mM phosphate buffer, pH 7.5, containing 154 mM NaCl and 1% BSA by incubating 100 µl of culture supernatant or ascitic fluid with 125I-hLHß[Leu119] (30,000 cpm) in a final volume of 200 µl. After incubation, the immune complexes were precipitated and counted as previously described (19). Direct binding studies with the various recombinant hLHß forms were achieved in a similar manner. Production of mAbs was then achieved by ip inoculation of nude mice with hybridoma cells.

Characterization of the antibody binding site
The precise location of the antigenic determinant recognized by the mAb selected in this study was deduced from results of hapten-inhibition experiments in ELISA and RIA tests. The antibody binding to the synthetic hLHß (104–119) peptide linked to a solid-phase support was inhibited by related peptides with shorter or longer amino acid sequences. The antibody dilution required for the competitive inhibition experiment was first determined on the precoated peptide. Displacement curves were then generated in the presence of increasing concentrations of the competitor, and residual antipeptide activity was measured (18). Competitive inhibition assays were also performed by RIA using a dilution of mAb that provided 50% maximal binding to 125I-labeled hLHß[Leu119]. Displacement curves were generated in the presence of increasing concentrations of synthetic peptides in binding experiments developed as previously described.

Gel electrophoresis and Western blotting
Preparations of recombinant carboxyl-terminus isoforms of hLHß were analyzed by SDS-PAGE under reducing conditions (5% ß-mercaptoethanol) on 12.5% acrylamide slab gels. Slab gels were run at 200 V for approximately 30 min. Proteins were then electrophoretically transferred to Immobilon polyvinylidene difluoride membranes (Millipore, St. Quentin, France) for 1 h at 250 mA and 20 C. They were detected using different monoclonal antipeptide antibodies. For this purpose, immunoblots were incubated for 1 h at room temperature in 0.1 M PBS, 0.2% Tween 20, and 5% delipidated milk, and, then, overnight in mAb-containing ascitic fluid diluted 1:100. After extensive washing, antibody-binding on the immunoblot was revealed by incubation for 2 h with a 125I-labeled sheep antimouse antibody (IM 1310, Amersham, Buckinghamshire, UK). After extensive washing, the immunoblot was dried and autoradiographied (Kodak film, Eastman Kodak, Rochester, NY).

Immunoradiometric assays
Two site "sandwich" monoclonal immunoradiometric assays (m-IRMA) were used to detect the hLHß isoforms present in various preparations. These assays are based on different antipeptide mAbs coated on a solid-phase support as capture antibodies, and on the 125I-labeled 518B7 monoclonal antibody as the radiolabeled indicator. Briefly, polystyrene beads (Precision Plastic Ball, Chicago, IL) were incubated overnight at 20 C in the presence of capture antibody (ascitic fluid diluted at 1:500) in 0.01 M phosphate buffer, 0.14 M NaCl, pH 7.2 (Pi/NaCl). Antibody-coated beads were incubated for 2 h with various biological preparations diluted in calf serum. After washing, hLHß isoforms captured on beads were detected by labeling with 125I-labeled 518B7 monoclonal antibody (100,000 cpm), diluted in Pi/NaCl containing 50% FCS, and incubated at room temperature for 1 h. After subsequent washing, the beads were counted. Nonspecific binding was determined by using calf serum.

Immunohistochemistry
Pituitary tissue was obtained at autopsy, fixed in formaldehyde 10%, and paraffin embedded. Four-micrometer sections were stained using Herlant’s tetrachrome, PAS-orange, and the Wilder technique to verify the absence of pituitary diseases. Immunohistochemistry tests were performed on serial sections using the indirect peroxydase method with nuclear hematein counterstaining. Briefly, immunocytochemical analysis was performed by incubating sections at 4 C overnight with either LHP03 or LHP09 (diluted 1:500). After subsequent washing, the Avidin Biotin Complex Vectastain kit (Vector Labs, Burlingame, CA) was used for staining and revelation was obtained with diaminobenzidine.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Production and characteristics of monoclonal antipeptide antibody LHP09
Cell fusions were performed with the splenocytes of two BALB/c mice immunized with the peptide-carrier conjugate. From 357 hybridomas, five antibody-secreting hybridomas were selected for their binding activity in the RIA test using the 125I-labeled hLHß[Leu119] preparation. The hybridoma which secreted antibody displaying the highest percentage of binding to 125I-hLHß[Leu119] (~35%) was selected, and the corresponding cells were cloned. The monoclonal antipeptide IgG1 antibody produced by these cells was designated LHP09. Ascitic fluids were obtained after ip inoculation of hybridomas into nude mice and were then tested in an RIA for their binding activity toward the different preparations of recombinant hLHß. Table 2Go shows the binding activities of monoclonal antipeptide antibodies LHP09 and LHP03 to the various radiolabeled molecular forms of hLHß. Antibody LHP03 that binds to the hLHß(43–52) region displayed identical binding to all forms of recombinant subunits. Interestingly, LHP09 reacted predominantly with the recombinant hLHß[Leu119] form, whereas its binding to hLHß[Phe120] was negligible.


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  		Table 2. Binding of monoclonal antipeptide antibodies LHP09 and LHP03 to the different recombinant hLHß isoforms

 
Characterization of the LHP09 antibody binding site
The location of the antigenic determinant recognized by LHP09 on hLHß was deduced from hapten inhibition experiments using various synthetic peptides, including hLHß (104–116), (104–119), (104–120), and (104–121) peptides and the composite hLHß(43–52)-(110–117) peptide, as competitors in both ELISA and RIA tests. Results from RIA experiments are presented in Fig. 1Go. We first determined the dilution of LHP09 which provided 50% maximal binding to 125I-hLHß[Leu119] (Fig. 1AGo). Displacement curves, generated in the presence of increasing concentrations of synthetic peptides, showed that only the hHLß (104–119) peptide exhibited striking potency in displacing bound 125I-hLHß[Leu119] from antibody LHP09, with a half-maximal inhibitory dose of 9 x 10-7 M. All the other peptides corresponding to the various carboxyl-termini of hLHß had no effect (Fig. 1BGo). As suggested by the direct binding study, LHP09 appeared to bind selectively to the [Leu119] carboxyl terminal part of the hLHß subunit. Similar hapten inhibition experiments were performed with LHP09 by ELISA. Results were in total agreement with those observed by competitive inhibition RIA (data not shown).



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  		Figure 1. Characterization of the LHP09 binding site. A, Binding of mAb LHP09 to 125I-labeled hLHß[Leu119] and (B) inhibition of 125I-labeled hLHß[Leu119] binding to mAb LHP09 by synthetic peptides mimicking various hLHß carboxyl-terminus: ß(104–116; {triangleup}), ß(104–119; {circ}), ß(104–120; {square}), and ß(104–121; {diamond}). Dose-response curves were generated as described in Materials and Methods, in the presence of increasing concentrations of competitor.

 
Western blotting of recombinant hLHß forms
Immunoblots were performed under reducing conditions on the various preparations of recombinant hLHß[Ser116], hLHß[Leu119], hLHß[Phe120] and hLHß[Leu121]. Results presented in Fig. 2Go showed that the LHP09 antibody exhibited a strong signal with the preparation corresponding to hLHß[Leu119] and no detectable response with the other recombinant hLHß subunits. A monoclonal antipeptide antibody, designated LHP10 (Robert, P., J. Pantel, F. Troalen, D. Bellet, and J. R. Bidart, manuscript in preparation), reacting with the region encompassing the 110–121 portion of hLHß and used as a control, immunostained all the four recombinant hLHß forms. These observations confirmed that monoclonal LHP09 antibody preferentially recognizes the hLHß[Leu119] form.



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  		Figure 2. Western immunoblotting of various recombinant hLHß using either mAb LHP09 or mAb LHP10. Gel electrophoresis, Western blotting, and immunodetection were performed as described in Materials and Methods.

 
Characterization of the hLHß[Leu119] form in hLH
To investigate the presence of hLHß[Leu119] in hLH prepared from different natural sources, we constructed two-site m-IRMAs based on various monoclonal antipeptide antibodies as capture mAbs: 1/mAb LHP09 was used to capture the hLHß[Leu119] form specifically; 2/mAb LHP03, which is directed to the hLHß(43–52) region, binds to both the hLH and the free hLHß subunit; and 3/mAb LHP04 recognizes the hLHß(110–117) portion and binds only to free hLHß. All these assays used 125I-518B7 as the tracer mAb. Figure 3AGo shows that the recombinant hLHß[Leu119] form was detected by the three m-IRMAs. Analysis of the purified hLHß (AFP-3282B), resulting from the dissociation of pituitary hLH, indicated that this preparation contains a significant amount (about 5–8% of total hLHß) of the hLHß[Leu119] form (Fig. 3BGo). In contrast, LHP03 bound only to the dimeric hLH (AFP-8270B), whereas no significant binding was observed with LHP09 and LHP04 mAbs used as capture mAbs (Fig. 3CGo). To investigate this observation further, we used heat treatment of hLH that is known to dissociate the dimeric glycoprotein hormone into its free or uncombined {alpha}- and ß-subunits. After heating at 56 C for 1 h, the binding activity of the final product was then measured by the m-IRMAs. Results presented in Fig. 3DGo show that heat-dissociated hLH, corresponding to the uncombined ß-subunit, was detectable in the m-IRMAs based on LHP03, LHP04, and LHP09. Taken together, these results demonstrate that the hLHß[Leu119] form is present in natural pituitary hLH and that LHP09 and LHP04 antibodies bind only to the free hLHß-subunit.



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  		Figure 3. Binding activities of recombinant hLHß[Leu119] isoform (A), pituitary hLHß (B), pituitary hLH (C), and heat-treated pituitary hLH (D) using m-IRMAs based on different antipeptide mAbs as capture antibodies and mAb 125I-518B7 as the tracer: LHP03-125I-518B7 ({diamond}), LHP04-125I-518B7 ({circ}), LHP09-125I-518B7 ({square}). MAb LHP03 reacts with the hLHß(43–52) region, accessible on both hLH and the free ß-subunit; mAb LHP04 binds to the hLHß(110–117) region, only accessible on the free hLHß; and mAb LHP09 specifically binds to the free hLHß[Leu119] isoform.

 
To check whether the hLHß[Leu119] form is detectable in urine from individuals, we performed similar experiments using a commercial preparation of urinary hLH submitted or not to thermal dissociation. Intact urinary hLH exhibited a weak but significant signal in the m-IRMA based on LHP04, indicating the presence of free hLHß in urine, whereas no signal was detectable in the m-IRMA based on LHP09 (Fig. 4AGo). After heat-treatment, similar curves were obtained with m-IRMAs based on LHP03 and LHP04, suggesting that the whole dimer had been dissociated (Fig. 4BGo). However, a negligible signal was obtained when LHP09 was used as the capture mAb in the m-IRMA. These observations suggest that the hLHß[Leu119] form is not present in urine from individuals.



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  		Figure 4. Binding activity of hLH prepared from urines of postmenopausal women before (A) and after heat-treatment (B) in m-IRMAs developed as described in Fig. 4Go.

 
Immunohistochemical detection of hLHß[Leu119] in the pituitary gland
Immunohistochemical analysis with antibodies LHP03 and LHP09 was used to investigate the expression of the hLHß[Leu119] form in the pituitary gland (Fig. 5Go, A and B). The two mAbs immunostained particular cells that appeared to be scattered throughout the anterior pituitary, in agreement with gonadotroph cell distribution. Some differences were observed in the pattern of cellular immunostaining: a weaker signal was observed with LHP09 and this antibody displayed preferential staining under the plasmic membrane. In contrast, LHP03 immunostained the cells completely.



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  		Figure 5. Expression of the hLHß[Leu119] isoform in a normal adult pituitary as immunostained by mAb LHP09 (A). Cells expressing hLH were stained by LHP03 (B). Magnification, x250.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Human lutropin exhibits molecular heterogeneity that affects both its biological potency and the duration of its activity (20). Variations in gonadotropin molecular weight are mainly attributed to highly variable carbohydrate structures, and oligosaccharide heterogeneity has been widely investigated in naturally occurring hLH isoforms as well as in recombinant hLH (21). Analysis of hLH peptidic heterogeneity indicated that subtle changes can occur at the primary structure level. Allelic variation at residues Trp8 and Ile15 has been recently described (22), and this may affect both the bioactivity and the turnover rate of the hormone (23). Amino-terminal heterogeneity has been observed in LHß subunits from only two species but never in human hLHß (7). Proteolytic cleavage was commonly observed in hLH preparations around the ß45–50 region, and the potency of the nicked hormones appears to be reduced (5, 6). Carboxyl terminal heterogeneity has been reported for hLHß as well as for the ß-subunit of other mammalian lutropins. The initial sequence studies on hLHß provided discordant results on the carboxyl terminus which appeared to end at either position Gln114 (8) or Gly117 (9). Moreover, cloning of the hLHß gene indicates that it can encode a protein composed of 121 amino acid residues (11). This discrepancy at the primary structure level between protein sequencing and gene cloning has also been described for the ß-subunit of hTSH (24).

To obtain information on the carboxyl terminus heterogeneity of the hLH ß-subunit in naturally occurring hLH forms, we designed a strategy based on the selection of a monoclonal antibody directed against a synthetic peptide mimicking the hLHß (104–119) region. This antipeptide mAb, designated LHP09, was proven to be specific for hLHß ending at position Leu119 as it displayed negligible binding to other forms of the ß-subunit presenting different carboxyl terminal residues. The antigenic region recognized by this antibody likely encompasses residues Cys110 to Leu119. In effect, Cys110 is engaged in a disulfide bond with Cys26 (25) and linear epitopes usually comprise 7–9 amino acid residues. Interestingly, the epitope recognized by LHP09 was not accessible to antibody binding to intact dimeric hLH. This observation suggests that the hLHß (104–119) region is probably masked by the vicinity of the {alpha}-subunit on the dimer, as suggested by the three-dimensional structure of hCG (25). Alternatively, the conformation of hLH and free hLHß might be different in this region.

A two-site "sandwich" immunoassay was then developed to investigate the presence of the hLHß[Leu119] form in different preparations of hLH. Our results indicate that this particular form is present at the pituitary level and absent in a preparation originating from urine of postmenopausal women. As it is difficult to obtain sufficient amounts of seric hLH, its presence in blood remains to be investigated. Taken together, these observations suggest that the hLHß[Leu119] form may constitute a transitional form in the biosynthesis of mature hLH. Indeed, in the anterior pituitary gland, glycoprotein hormones undergo complex posttranslational processing (26, 27). It has been postulated that, on hTSH and hLH, the peptidic extensions predicted from complementary DNAs, which are longer than that found on the mature proteins, may be responsible for the retention of these free ß-subunits by the endoplasmic reticulum (28). A recent study indicated that the hydrophobic hLHß heptapeptide stretch plays a critical role in both posttranslational processing of the ß-subunit and in its efficiency when combining with the {alpha}-subunit to form the biologically active dimer hLH. Indeed, in in vitro experiments, hCGß, which contains a hydrophilic 31-amino acid extension, is efficiently secreted, whereas secretion and assembly of hLHß are inefficient (12). However, a recent report indicated that the presence of the hLHß heptapeptide stretch on the secreted forms does not appear to influence the binding activity (29). Although we cannot totally rule out that heterogeneity on the carboxyl terminus simply represent random cleavage, our observations suggest that, during its biosynthesis, hLHß is processed as several intermediary forms and that the hydrophobic hLHß heptapeptide stretch is not present on the mature secreted hLH.

Immunological probes directed to the various carboxyl termini of the hLHß subunit may constitute powerful tools for rapid and sensitive detection of the molecular forms of this hormone produced in different physiopathological conditions or by recombinant DNA technology. For example, clinically nonfunctioning pituitary adenomas, which represent about a quarter of all pituitary tumors, do not secrete gonadotropins even though the majority of these adenomas are gonadotropinomas, as demonstrated by immunohistochemical methods. It is not clear whether this absence of secretion is due to aberrant molecules and/or to abnormal processing of the hormones. The recent availability of recombinant gonadotropins is a major advance in reproductive medicine (30, 31). Indeed, there are several advantages to these preparations compared with those obtained by extraction from urine: 1) they reduce the risks of human tissue-derived pathogen contaminants; 2) the availability and consistency of the product is greater; and 3) the spectrum of therapeutic possibilities is expanded (32, 33). Various experimental protocols are used to characterize recombinant hormones in detail (32, 34). At the peptidic level, analysis of the primary structure of recombinant products focuses on amino-terminal heterogeneity. Immunochemical detection based on specific antibodies is a rapid and convenient way to investigate carboxyl-terminal heterogeneity. Furthermore, these antibodies can also be used to separate a particular form present in a spectrum of isoforms completely. Finally, these reagents may also help to characterize of reliable standards needed to improve the comparability of immunoassays and bioassays (35, 36).


   Acknowledgments
 
We are indebted to Drs. M. Dreano and A. Ythier for providing us with recombinant molecular forms of hLHß and for helpful discussions. We also thank J. L. Bobot and J. P. Levillain for expert technical assistance in the production of antibodies and in the synthesis of peptides, respectively, and L. Saint-Ange for correcting the manuscript.


   Footnotes
 
1 This work was supported by grants from ATT-Serono GF5636 and from the Interface Chimie-Biologie (CNRS/ARC). In this paper, the human sequence is used as a reference. Back

Received June 11, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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A. Jablonka-Shariff, C. A. Pearl, A. Comstock, and I. Boime
A Carboxyl-terminal Sequence in the Lutropin {beta} Subunit Contributes to the Sorting of Lutropin to the Regulated Pathway
J. Biol. Chem., April 25, 2008; 283(17): 11485 - 11492.
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