Indian J Exp Biol

APRIL 2002

CODEN: IJEB (A6)  40(4)  377-506  (2002)

ISSN: 0019-5189

 

 

 

Indian Journal of Experimental Biology

http : // www.niscom.res.in; http : // www.bioline.org.br/

 

VOLUME 40

NUMBER 4

APRIL 2002

 

CONTENTS

Symposium in Print on Structure and Function of Gonadotropins

 

Human follitropin: Structure-activity relationships

James A Dias

385

 

 

 

 

 

Functional correlates of the growth factor structure of pituitary follitropin

M R Sairam

393

 

 

 

 

Structural studies of hLH/CG receptor extracellular binding domain

Leslie I Lobel, Susan Pollak & Joyce W Lustbader

402

 

 

 

 

Fusion proteins of human gonadotropins and gonadotropin-receptor complexes

David Puett & Prema Narayan

415

 

 

 

 

Interaction and activation of luteinizing hormone receptor

Yong Sang Song, Chong Seoung Yi, Chang Woo Lee, Myung Kun Jeoung,

Ki Sung Ryu, Inhae Ji & Tae H Ji

 

 

424

 

 

 

 

Structure function analysis: Lessons from human chorionic gonadotropin

Ashish Banerjee, Natarajan Venkatesh & G Satyanarayana Murthy

434

 

 

 

 

Human chorionic gonadotropin : An update on its receptor binding regions

K Sankaranarayana Iyer, Ashwini Kumar Mishra, Smita D Mahale &

Dhanashree D Jagtap

448

 

 

 

 

Studies on chemical modification of ovine luteinizing hormone and its subunits

with different heterobifunctional cross-linking agents

Ranjit C Singh, Vinod Singh & Anis Alam

 

456

 

 

 

 

Role of disulphide bond formation in folding, secretion, and assembly of human

chorionic gonadotropin subunits

Elliott Bedows, Ryan J Darling, Jason A Wilken &  Simon A Sherman

 

467

 

 

 

 

Design and synthesis of hormonotoxin for selective targeting of gonadal cells

Anis Alam, Ranjit C Singh & Vinod Singh

477

 

 

 

 

Equine gonadotropins as models for glycosylation studies

George R Bousfield & Vladimir Y Butnev

486

 

 

 

 

 

 

 

 

 

 

 

 

 

Indian Journal of Experimental Biology

Vol. 40, April 2002, pp. 385-392

 

 

 

Human follitropin: Structure-activity relationships

James A Dias

Wadsworth Center, David Axelrod Institute of Public Health, Division of Molecular Medicine,
New York State Departmrny of Health, Albany, NY 12208

Department of Biomedical Sciences, School of Public Health, University at Albany, New York

Email: James.Dias@Wadsworth.org

Studies of human follitropin (hFSH) structure-activity relationships from the author’s laboratory are reviewed. These include mutagenesis studies that complemented the determination of the three dimensional structure of hFSH. Despite a large extracellular domain of the FSH receptor and the complexity and size of both the receptor and the hFSH molecule, only a handful of hFSH amino acids are required for high affinity interaction of hFSH with its receptor. These amino acids, contributed by both alpha and beta subunit chains, cluster together in a discrete location, brought together by protein folding and subunit association. Specificity of binding is enabled by the beta subunit, but both subunits contribute to high affinity interactions with receptor. Structural differences exist between the three dimensional structures of glycosylated hFSH and deglycosylated placental glycoprotein hormone. Carbohydrate seems unlikely to play a major role in protein structure, ending speculation that glycosylation-dependent protein conformation is the key to signal transduction.

 

 

 

Indian Journal of Experimental Biology

Vol. 40, April 2002, pp. 393-401

 

 

Functional correlates of the growth factor structure of pituitary follitropin

M R Sairam

Molecular Reproduction Research Laboratory, Clinical Research Institute of Montreal, Quebec, H2W1R7 Canada

Tel.: (514) 987-5582; Fax: (514) 987-5585; e-mail: sairamm@ircm.qc.ca

Pituitary follitropin (FSH) is an important member of the glycoprotein hormone family that is required for maintenance of reproductive functions. The hormone has been visualized as having motifs such as the cystine knots that are typical of other growth factors with mitogenic functions. By studying its receptor system in the ovary and testis, we have demonstrated the existence of the FSH receptor that has a single transmembrane domain. This receptor functions via a non-cyclic AMP dependent pathway and stimulates Ca2+ mobilization and activation of extracellular activated kinases. Thus its actions are separate from the classical Gs coupled receptor. This constitutes the first functional evidence for the growth factor structure of the hormone. Alternative splicing of a single large FSH-R gene produces all the FSH receptor variants. The polymorphic forms of the hormone might interact differently with these receptors to influence target cell growth patterns and steroidogenic responses.

 

 

 

Indian Journal of Experimental Biology

Vol. 40, April 2002, pp. 402-414

 

 

Structural studies of hLH/CG receptor extracellular binding domain

Leslie I Lobel, Susan Pollak & Joyce W Lustbader

Center for Reproductive Science and Department of Obstetrics and Gynecology,
Columbia University, 630 West 168th. St., New York, N.Y. 10032

The structure of the hormone-receptor interface for hLH/CG receptor was examined to identify potential small molecule antagonists or agonists that act on the receptor. Two parallel yet complementary sets of experiments were initiated. Firstly, the production and purification of large quantities of the extracellular binding domain of hLH/CG receptor to be used for crystallographic studies. Preparative quantities of properly folded extracellular domain of hLH/CG receptor as a  fusion protein in a prokaryotic based system is being utilized for growing crystals for diffraction studies. The second approach involves a small molecule screen based analysis of the binding domain of hLH/CG receptor. This technique is being utilized to map the molecular surface of the receptor-ligand interface of hLH/CG receptor by identifying specific small molecule antagonists and/or agonists, defining the functional moieties responsible for their activities and examining the bound or “docked” conformation of these molecules with the receptor. These studies offer a more rapid analysis of the receptor-ligand interface and identification of small molecules that act on the receptor. The progress with these latter studies and the potential of this approach are discussed in detail in this review.

 

 

Indian Journal of Experimental Biology

Vol. 40, April 2002, pp. 415-423

 

 

 

Fusion proteins of human gonadotropins and gonadotropin-receptor complexes

David Puett* & Prema Narayan

Department of Biochemistry & Molecular Biology, Life Sciences Building, University of Georgia, Athens, Georgia 30602, USA

Three glycoprotein hormones comprise the family of gonadotropins in humans, luteinizing hormone (LH), chorionic gonadotropin (CG), and follicle-stimulating hormone (FSH), composed of a common a subunit and a hormone-specific b subunit. These heterodimers act via two G protein-coupled receptors, the LH receptor (LHR) and the FSH receptor (FSHR), that are expressed on gonadal cells. The b subunits of LH and CG are sufficiently similar that the two holoproteins bind to and activate a common receptor, LHR. Using protein engineering, several laboratories, including ours, have designed and expressed bioactive fusion proteins of the two subunits of human CG, LH, and FSH. Most of these have been of the form, N-b-a-C, although the reverse configuration, N-a-b-C, has been shown to be active as well. Complementing this approach, bioactive intersubunit disulfide-linked gonadotropins, i.e. a-S-S-b, have also been designed and expressed. Site-directed mutagenesis has been used to prepare a number of altered forms of the single chain and intersubunit disulfide-linked gonadotropins, including single amino acid residue replacements, deletions, elimination of N-linked glycosylation sites, and elimination of disulfides. In many cases it was found, quite surprisingly, that the properties of the mutant fusion and intersubunit disulfide-linked gonadotropins were not predictable from the known characteristics of the same alterations of the individual subunits. Thus, such complexes provide unique reagents for research and eventual clinical utilization. Extending the studies on fusion gonadotropins, our laboratory has designed and expressed yoked or single chain human CG-LHR complexes such as N-a-CGb-LHR-C and N-CGb-a-LHR-C, with the carboxy-terminal peptide of CGb serving as a linker. These membrane-associated fusion proteins lead to constitutive receptor activation as judged by extraordinarily high levels of cellular basal cAMP in transfected cells. This paper reviews the myriad forms of single chain gonadotropins, single chain gonadotropin-receptor complexes, and intersubunit disulfide-linked gonadotropins that have recently been described.

 

 

Indian Journal of Experimental Biology

Vol. 40, April 2002, pp. 424-433

 

 

Interaction and activation of luteinizing hormone receptor*

Yong Sang Song, ChongSeoung Yi, ChangWoo Lee, MyungKun Jeoung, KiSung Ryu, Inhae Ji & Tae H Ji#

Department of Chemistry, University of Kentucky, Lexington, KY 40506-0055

The luteinizing hormone receptor is a G protein coupled receptor with unique structural and functional features, consisting of two halves. The N-terminal extracellular half (exodomain) binds the hormones, whereas the C-terminal membrane associated half (endodomain) is responsible for receptor activation. This article describes the sequence of the interactions among human chorionic gonadotropin, the exodomain and endodomain. For example, the hormone binds initially to the exodomain, the resulting hormone/exodomain complex modulates the interaction between the exodomain and endodomain, and this ternary complex is involved in signal generation. This sequence suggests that the exodomain and endodomain are intimately associated prior to the hormone binding, contrary to the view that the exodomain and endodomain are indepen­dent at least functionally.

 

 

 

Indian Journal of Experimental Biology

Vol. 40, April 2002, pp. 434-447

 

 

Structure function analysis: Lessons from human chorionic gonadotropin

Ashish Banerjee, Natarajan Venkatesh & G Satyanarayana Murthy*

Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560 012, India

Human chorionic gonadotropin (hCG) belongs to the family of cystine knot growth factors secreted by the placenta very early during pregnancy. Owing to the complexity of its structure and by virtue of being a heterodimer, it has proven to be an excellent model system for the study of protein folding and protein-protein interaction. Several groups including us have carried out structure function analysis of hCG and related glycoprotein hormones in consideration of the importance of these hormones in reproduction. In this article we have tried to highlight some of the recent work reported in literature in the backdrop of some classical work before the crystal structure was reported. We have also tried to bring out the apparent satire in the whole story, which obviously to us, is the failure to obtain a small molecular weight substitute for the hormone.

 

 

Indian Journal of Experimental Biology

Vol. 40, April 2002, pp. 448-455

 

 

Human chorionic gonadotropin : An update on its receptor binding regions

K Sankaranarayana Iyer*, Ashwini Kumar Mishra, Smita D. Mahale & Dhanashree D. Jagtap

Peptide and Protein Research Laboratory, Institute for Research in Reproduction (ICMR), Mumbai  400 012, India

Human chorionic gonadotropin (hCG), an important member of the glycoprotein hormone family, plays a crucial role in the establishment and maintenance of pregnancy. Glycoprotein hormones are all heterodimers composed of a common a-subunit and a hormone-specific b-subunit noncovalently linked. These hormones exert their action by binding to specific receptors on the target cells. As both the subunits are involved in receptor binding, the binding sites on the hormones have to be topographical extending across both subunits. This article attempts to critically review the work on the receptor binding regions of hCG in the a- and b-subunits. A variety of approaches like chemical modification, site-directed mutagenesis, hormone chimeras and synthetic peptides have been used to map the receptor binding regions of hCG. There is agreement on the involvement of a number of regions in the a- and b-subunits in receptor binding but, there are also some contrasting observations. The regions 30-45 and 81-92 in the a-subunit appear to be involved in receptor binding whereas 8-22, 85-95, 93-100 and 100-110 are the consensus receptor binding regions in the b-subunit. Using disulphide peptides of the b-subunit as probes, recently the regions around Cys(9-57) and Cys(23-72) disulphide bonds have been identified to be important for receptor binding. After sifting through the available data two potential receptor binding sites in the b-subunit have been proposed.

 

 

 

Indian Journal of Experimental Biology

Vol. 40, April 2002, pp. 456-466

 

 

Studies on chemical modification of ovine luteinizing hormone and its subunits with
different heterobifunctional cross-linking agents

Ranjit C Singh, Vinod Singh* & Anis Alam

Hormone Biochemistry Laboratory, Institute of Self-Organising Systems and Biophysics, and Department of Biochemistry (AA),
North-Eastern Hill University, Permanent Campus, Shillong 793 022, India

Received 15 December 2001

Increasing use of heterobifunctional cross-linking agents (HBCLA) in the design of defined conjugates for selective targeting and inducing immune response and to understand the significance of chemical modification of -NH2 group of lysine residues of native oLH and its a- and b- subunits of ovine luteinizing hormone (oLH) on, immunological, biological activity of the hormone and a:b subunit recombination, the e-NH2 group(s) of oLH and its a- and b- subunits were separately and sequentially modified with different heterobifunctional cross-linking agents (HBCLA) such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl 6-[3-(2-pyridyldithio)propionamido] hexanoate (LC-SPDP), 2-iminothiolane (2IT), and 4-succinimidyloxcarbonyl-a-methyl-a(2-pyridyldithio)toluene (SMPT). The aoLH modified by HBCLA recombine to native boLH was judged by reverse phase high performance chromatography (RP-HPLC) analysis. Similarly boLH modified by HBCLA also recombine to native aoLH. The sequential modification of subunits led to progressive reduction in immunoreactivity, receptor binding and steroidogenic activity of the dimer. The modification of six or more e-NH2 groups in aoLH although recombine fully with native boLH but failed to show receptor binding, anti-oLH antibody reactivity and steroidogenic activity. The modification upto four groups in aoLH compromised immunological and biological activities but further addition of two or more groups completely abolished immunological and biological activity of the recombinants indicating the importance of later two amino groups in the receptor binding and steroidogenic activity.

 

 

Indian Journal of Experimental Biology

Vol. 40, April 2002, pp. 467-476

 

 

Role of disulphide bond formation in folding, secretion, and assembly of
human chorionic gonadotropin subunits

Elliott Bedows1,2,3,4*, Ryan J Darling1,2†, Jason A Wilken1,3 & Simon A Sherman1

1The Eppley Institute for Research in Cancer and Allied Diseases

2Department of Pharmacology, 3Department of Biochemistry and Molecular Biology, 4Department of Obstetrics and Gynecology

University of Nebraska Medical Center, Omaha, NE 68198, USA

Received 15 December 2001

Ability of a glycoprotein hormone to fold and assemble correctly is an essential requirement for the attainment of its biological activity. Cells have a quality control system to assure that when proteins of the endoplasmic reticulum misfold or fail to assemble correctly they do not exit the cell. But human chorionic gonadotropin (hCG) is an exception to this rule as is illustrated by the following observations. First, both hCG subunits are secreted efficiently as unassembled monomers. Second, some mis(un)folded forms of hCG-b can be efficiently secreted from cells that do not facilitate the secretion of other misfolded proteins. Third, the efficient secretion of an assembly incompetent a-subunit occurs in spite of lacking both of its non-cystine knot disulphide bonds. And fourth, hCG heterodimers that contain misfolded a-subunits as a result of lacking cystine knot disulphides are also secreted. In this report, we review direct experimental evidence demonstrating that the structural requirements necessary for the secretion of hCG subunits differ from the structural requirements necessary for hCG assembly by using disulphide bond formation as an index with which to monitor the folding, assembly, and secretion of hCG and its a- and b-subunits.

 

Indian Journal of Experimental Biology

Vol. 40, April 2002, pp.

 

Design and synthesis of hormonotoxin for selective
targeting of gonadal cells

Anis Alam, Ranjit C. Singh & Vinod Singh*

*Hormone Biochemistry Laboratory, Institute of Self-Organising Systems and Biophysics, and Department of Biochemistry (AA),
North-Eastern Hill University, Permanent Campus, Shillong 793 022, India

 

With the aim of developing a cytotoxic agent that could selectively target the appropriate steroid producing cells in the gonads, hormone-toxin conjugates were synthesized with the use of different heterobifunctional cross linking agents (HBCLA) such as, 2-iminothiolane HCl (2IT), N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP, 8.6 A) and N-succinimidyl 6-[3-(2-pyridyldithio) propionamido] hexanoate (LC-SPDP, 15.6 A). A complete physico-chemical, immunochemical and biochemical analysis was performed. The linkage occurred through the e-NH2 groups of a-subunit of oLH as analysed by RP-HPLC analysis. But it was difficult to ascertain the site of linkage in hCG-gelonin conjugate. A 1:1 (hormone:gelonin) molar ratio was obtained when determined with spectrophotometric, gel electrophoresis and amino acid composition methods. The competitive displacement analysis indicates that the binding occurs via the hormone part and leaving gelonin free which was probed with the gelonin antibodies. The conjugates exhibited comparable immunoreactivity but the receptor binding and cytotoxicity of the conjugates prepared with 2IT were higher than the hormonotoxin prepared with the use of SPDP or LC-SPDP. Therefore, it is concluded that higher receptor binding and cytotoxicity may be due to the retention of positive charge on the lysine residues of oLH, which was preserved during the conjugation process. However, the cytotoxicity of oLH based hormonotoxins remained unaffected with the use of long chain spacer arm that is believed to be used generally to avoid steric hindrance. Based on the data presented here, it may be concluded that it is possible to synthesize oLH or hCG based hormonotoxins which specifically interacted with LH/hCG receptor and significantly inhibited protein synthesis in the tumor cells bearing receptors for gonadotropins. The detailed in vivo experiments under investigation would further demonstrate their effectiveness. If successful, a new class of luteolytic may be available in future.

 

Indian Journal of Experimental Biology

Vol. 40, April 2002, pp. 486-506

 

Equine gonadotropins as models for glycosylation studies

George R Bousfield & Vladimir Y Butnev

Department of Biological Sciences, Wichita State University, Wichita, KS 67260, USA

Tel : 001-316-978-6088; Fax:  001-316-978-3772; email: george.bousfield@wichita.edu

Gonadotropins require carbohydrate for their normal physiological function, however, determining the mechanisms involved is complicated by the existence of several glycosylation sites decorated with a variety of oligosaccharide structures that contribute to various aspects of gonadotropin structure and function. Partially overlapping functions of these oligosaccharides provide additional complications. N-linked oligosaccharides contribute to protein folding, heterodimer stability, circulatory survival, receptor-binding affinity, and cellular activation. O-linked oligosaccharides influence N-linked oligosaccharide processing and extend circulatory survival. Equine LH and eCG provide useful subjects for studying the influence of carbohydrate on gonadotropin action because they differ only in their carbohydrate moieties, which produce substantial effects on their activities. Moreover, because these hormones bind FSH receptors, as well as LH receptors, they can be used to study the role of carbohydrate in hormone activation of both receptors.