William R. Moyle

Quantitative explanation for increased affinity shown by mixtures of monoclonal antibodies: importance of a circular complex.

Mixtures of some but not all monoclonal antibodies which bind to separate epitopes on human chorionic gonadotropin (hCG) show an increased affinity for the hormone. To find an explanation for the increase in affinity, we developed a mathematical model which predicts the quantities of intermediates formed when pairs of IgG1 mouse monoclonal antibodies having affinities of approximately 10(8) M-1 for hCG are mixed with the hormone. At low antibody concentrations (i.e. less than 1 nM or 0.15 micrograms/ml) analysis of possible antibody-hormone combinations, including linear and circular chains composed of less than 12 molecules of antibody and 12 molecules of hCG, suggests the increase in affinity is due to formation of a circular complex containing two molecules of antibody and two of hCG. Further, the model predicts that the circular complex will be the major species formed at antibody-antigen equivalence. This prediction is supported by experimental observations on the molecular weight of a new complex formed in the presence of hCG and the mixture of the monoclonal antibodies. In addition, based on experimental values of binding constants for individual antibodies to hCG, the model correctly quantifies the loss in complex observed in the presence of excess hCG antigen. At high antibody concentrations (i.e. greater than 10 nM or 1.5 micrograms/ml) the formation of linear chains of antibody hCG pairs becomes appreciable and contributes to the increase in apparent affinity of the mixture for hCG. These results suggest that the observed affinity of complex mixtures of antibody for antigens containing multiple epitopes calculated from Scatchard plots may not be related to the affinity or avidity of any of the antibody species for a given epitope.

Cloning of rat lutropin (LH) receptor analogs lacking the soybean lectin domain

cDNAs coding for rat ovarian luteinizing hormone receptor analogs lacking three of the leucine repeats were detected in a library which had been prepared from rat luteal tissue undergoing human chorionic gonadotropin-induced luteinization. These leucine repeats correspond to amino acids 206-267 and contain the portion of the receptor that is homologous to the soybean lectin. The cDNA library also contained a receptor analog lacking amino acids 321-700 which code for the transmembrane domain. S-1 mapping suggests that this latter form constitutes approximately half of all receptor-related mRNA.

Models of glycoprotein hormone receptor interaction

The glycoprotein hormones regulate reproduction and development through their interactions with receptors in ovarian, testicular, and thyroid tissues. Efforts to design hormone agonists and antagonists useful for treating infertility and hyperthyroidism would benefit from a molecular understanding of hormone-receptor interaction. The structure of a complex containing FSH bound to a fragment of its receptor has been determined at 2.9 Å resolution, but this does not explain several observations made with cell-surface G protein receptors and may reflect the manner in which FSH binds a short alternate spliced receptor form. We discuss observations that must be explained by any model of the cell-surface G protein-coupled glycoprotein hormone receptors and suggest structures for these receptors that satisfy these requirements. Glycoprotein hormones appear to contact two distinct sites in the extracellular domains of their receptors, not just the leucine-rich repeat domain. These dual contacts contribute to ligand binding specificity and appear to be essential for signal transduction. As outlined in this minireview, differences in the manners in which these ligands contact their receptors explain why some ligands and ligand analogs interact with more than one class of receptor and why some receptors and receptor analogs bind more than one ligand. The unique manner in which these ligands appear to interact with their receptors may have facilitated hormone and receptor co-evolution during early vertebrate speciation.

Threading of a glycosylated protein loop through a protein hole: implications for combination of human chorionic gonadotropin subunits.

Chorionic gonadotropin (hCG) is a heterodimeric placental glycoprotein hormone essential for human reproduction. Twenty hCG β‐subunit residues, termed the seatbelt, are wrapped around α‐subunit loop 2 (α2) and their positions “latched” by a disulfide formed by cysteines at the end of the seatbelt (Cys 110) and in the β‐subunit core (Cys 26). This unique arrangement explains the stability of the heterodimer but raises questions as to how the two subunits combine. The seatbelt is latched in the free β‐subunit. If the seatbelt remained latched during the process of subunit combination, formation of the heterodimer would require α2 and its attached oligosaccharide to be threaded through a small β‐subunit hole. The subunits are known to combine during oxidizing conditions in vitro, and studies described here tested the idea that this requires transient disruption of the latch disulfide, possibly as a consequence of the thioredoxin activity reported in hCG. We observed that alkylating agents did not modify either cysteine in the latch disulfide (Cys 26 or Cys 110) during heterodimer formation in several oxidizing conditions and had minimal influence on these cysteines during combination in the presence of mild reductants (1–3 mM β‐mercaptoethanol). Reducing agents appeared to accelerate subunit combination by disrupting a disulfide (Cys 93–Cys 100) that forms a loop within the seatbelt, thereby increasing the size of the β‐subunit hole. We propose a mechanism for hCG assembly in vitro that depends on movements of α2 and the seatbelt and suggest that the process of glycoprotein hormone subunit combination may be useful for studying the movements of loops during protein folding.

hCGβ Residues 94–96 alter LH activity without appearing to make key receptor contacts

The ability of human chorionic gonadotropin (hCG) to distinguish lutropin (LHR) and follitropin (FSHR) receptors is controlled principally by beta-subunit residues 94-117. To learn how residues 94-96 (Arg-Arg-Ser) influence LHR binding, we studied the effects of replacing them on the LH and FSH activities of a bifunctional hCG analog in which residues 101-109 were derived from FSH. Analogs containing 1-3 arginines and no aspartates at residues 94-96 bound LHR with 25-400% the potency of hCG. When residues 94-96 were neutral or contained 1-3 aspartates, LHR binding was reduced 6-100 fold but remained at least ten-fold greater than the negative control analog containing residues 94-117 derived from FSH. Residues 94-96 had little influence on FSHR binding. These observations support a model [Moyle et al. (1995) J. Biol. Chem. 270:20,020] in which residues 94-96 influence LHR binding specificity primarily through an effect on hormone conformation rather than by direct participation in essential high affinity receptor contacts.

Analysis of human choriogonadotropin core 2 o-glycan isoforms

Measurements of human choriogonadotropin (hCG) isoforms containing core 2 o-glycans may be useful for diagnosis of Down Syndrome pregnancies and trophoblastic disease. As shown here, this isoform is also present in pituitary extracts, early pregnancy urine, and urine of postmenopausal women. Although, measurements of hCG isoforms may be useful in several clinical settings, this remains to be determined due to the lack of suitable standards and the difficulties of comparing data obtained in different laboratories. Here, we report that monoclonal antibodies B152 and CTP104 recognize the third (Ser132) and fourth (Ser138) o-glycans, respectively, in the carboxyterminal portion of the hCG beta-subunit. The proximity of these sites prevents B152 and CTP104 from binding simultaneously to isoforms containing core 2 o-glycans. Unlike B152, which binds only the core 2 isoform, CTP104 recognizes both glycan moieties. By measuring hCG with CTP104 in the presence and absence of B152, one can quantify both isoforms using the same readily available standard.

Glycoprotein Hormone Assembly in the Endoplasmic Reticulum I. THE GLYCOSYLATED END OF HUMAN α-SUBUNIT LOOP 2 IS THREADED THROUGH A β-SUBUNIT HOLE

Glycoprotein hormone heterodimers are stabilized by their unusual structures in which a glycosylated loop of the α-subunit straddles a hole in the β-subunit. This hole is formed when a cysteine at the end of a β-subunit strand known as the “seatbelt” becomes “latched” by a disulfide to a cysteine in the β-subunit core. The heterodimer is stabilized in part by the difficulty of threading the glycosylated end of the α-subunit loop 2 through this hole, a phenomenon required for subunit dissociation. Subunit combination in vitro, which occurs by the reverse process, can be accelerated by removing the α-subunit oligosaccharide. In cells, heterodimer assembly was thought to occur primarily by a mechanism in which the seatbelt is wrapped around the α-subunit after the subunits dock. Here we show that this “wraparound” process can be used to assemble disulfide cross-linked human choriogonadotropin analogs that contain an additional α-subunit cysteine, but only if the normal β-subunit latch site has been removed. Normally, the seatbelt is latched before the subunits dock and assembly is completed when the glycosylated end of α-subunit loop 2 is threaded beneath the seatbelt. The unexpected finding that most assembly of human choriogonadotropin, human follitropin, and human thyrotropin heterodimers occurs in this fashion, indicates that threading may be an important phenomenon during protein folding and macromolecule assembly in the endoplasmic reticulum. We suggest that the unusual structures of the glycoprotein hormones makes them useful for identifying factors that influence this process in living cells.

Glycoprotein hormone assembly in the endoplasmic reticulum: IV. Probable mechanism of subunit docking and completion of assembly.

The unique structures of human choriogonadotropin (hCG) and related glycoprotein hormones make them well suited for studies of protein folding in the endoplasmic reticulum. hCG is stabilized by a strand of its β-subunit that has been likened to a “seatbelt” because it surrounds α-subunit loop 2 and its end is “latched” by an intrasubunit disulfide bond to the β-subunit core. As shown here, assembly begins when parts of the NH2 terminus, cysteine knot, and loops 1 and 3 of the α-subunit dock reversibly with parts of the NH2 terminus, cystine knot, and loop 2 of the hCG β-subunit. Whereas the seatbelt can contribute to the stability of the docked subunit complex, it interferes with docking and/or destabilizes the docked complex when it is unlatched. This explains why most hCG is assembled by threading the glycosylated end of α-subunit loop 2 beneath the latched seatbelt rather than by wrapping the unlatched seatbelt around this loop. hCG assembly appears to be limited by the need to disrupt the disulfide that stabilizes the small seatbelt loop prior to threading. We postulate that assembly depends on a “zipper-like” sequential formation of intersubunit and intrasubunit hydrogen bonds between backbone atoms of several residues in the β-subunit cystine knot, α-subunit loop 2, and the small seatbelt loop. The resulting intersubunit β-sheet enhances the stability of the seatbelt loop disulfide, which shortens the seatbelt and secures the heterodimer. Formation of this disulfide also explains the ability of the seatbelt loop to facilitate latching during assembly by the wraparound pathway.

Glycoprotein Hormone Assembly in the Endoplasmic Reticulum III. THE SEATBELT AND ITS LATCH SITE DETERMINE THE ASSEMBLY PATHWAY

Vertebrate glycoprotein hormone heterodimers are stabilized by a strand of their β-subunits known as the “seatbelt” that is wrapped around loop 2 of their α-subunits (α2). The cysteine that terminates the seatbelt is “latched” by a disulfide to a cysteine in β-subunit loop 1 (β1) of all vertebrate hormones except some teleost follitropins (teFSH), wherein it is latched to a cysteine in the β-subunit NH2 terminus. As reported here, teFSH analogs of human choriogonadotropin (hCG) are assembled by a pathway in which the subunits dock before the seatbelt is latched; assembly is completed by wrapping the seatbelt around loop α2 and latching it to the NH2 terminus. This differs from hCG assembly, which occurs by threading the glycosylated end of loop α2 beneath the latched seatbelt through a hole in the β-subunit. The seatbelt is the part of the β-subunit that has the greatest influence on biological function. Changes in its sequence during the divergence of lutropins, follitropins, and thyrotropins and the speciation of teleost fish may have impeded heterodimer assembly by a threading mechanism, as observed when the hCG seatbelt was replaced with its salmon FSH counterpart. Whereas wrapping is less efficient than threading, it may have facilitated natural experimentation with the composition of the seatbelt during the co-evolution of glycoprotein hormones and their receptors. Migration of the seatbelt latch site to the NH2-terminal end of the β-subunit would have facilitated teFSH assembly by a wraparound mechanism and may have contributed also to its ability to distinguish lutropin and follitropin receptors.

Influence of Subunit Interactions on Lutropin Specificity IMPLICATIONS FOR STUDIES OF GLYCOPROTEIN HORMONE FUNCTION

Bovine lutropin (bLH) and human chorionic gonadotropin (hCG) are heterodimeric glycoprotein hormones required for reproduction. Both bind rat LH receptors (rLHRs), but hCG binds human LH receptors (hLHRs) 1000-10,000 fold better than bLH. We tested the premise that this difference in affinity could be used to identify lutropin receptor contacts. Heterodimers containing hCG/bLH α- or β-subunit chimeras that bound hLHR like hCG (or bLH) were expected to have hCG (or bLH) residues at the receptor contact sites. Analogs containing one subunit derived from hCG bound hLHR much more like hCG than bLH, indicating that each bLH subunit contains all the residues sufficient for high affinity hLHR binding. Indeed, the presence of bovine α-subunit residues increased the activities of some hCG analogs. The low hLHR activity of bLH was due primarily to an interaction between its α-subunit and β-subunit residue Leu95. Leu95 does not appear to contact the hLHR since it did not influence the hLHR activity of heterodimers containing human α-subunit. These observations show that interactions within and between the subunits can significantly influence the activities of lutropins, thereby confounding efforts to identify ligand residues that contact these receptors.