Human chorionic gonadotropin : Recombinant DNA expression and structure-function relationship studies

Abstract

Human Chorionic Gonadotropin (hCG) is a member of the family of glycoprotein hormones, which also includes the pituitary-derived Thyroid Stimulating Hormone (TSH), Follicle Stimulating Hormone (FSH), and Luteinizing Hormone (LH). These hormones are heterodimers composed of an identical subunit noncovalently associated with the hormone-specific subunit. Both subunits are products of different genes, synthesized separately, and associate noncovalently in the endoplasmic reticulum early in their secretory pathway. Formation of the heterodimer, accompanied by several conformational changes, is a prerequisite for generation of biologically active forms of these hormones.

Both the and subunits are N-glycosylated, with the hCG subunit having an additional four O-linked oligosaccharides located at the carboxyl-terminal extension of the polypeptide. N-linked glycosylation during biosynthesis facilitates protein folding and conformational maturation of the subunits into an assembly-competent, biologically active form. Further, the and subunits of these hormones contain 5 and 6 disulfide bonds, respectively, and the crystal structure of hCG indicates that both subunits belong to the Cystine knot family of proteins.

hCG has proved to be an important model for structure–function relationship studies of complex dimeric glycoproteins. Folding during biosynthesis of the subunits, elucidation of the role of glycosylation in the folding pathways in vitro and in vivo, as well as identification of domains important for subunit association, receptor binding, and signal transduction, have been the topics of active investigation. In addition, hCG has been reported to have immunocontraceptive potential. Active and passive immunization with hCG in primates and humans resulted in termination of pregnancy, forming the basis of hCG-based contraceptive vaccines.

A major limitation in the development of contraceptive vaccines is the lack of adequate quantities of hormonal antigen free of pathological elements. The objective of the present study was hyperexpression of glycosylated, properly folded, and biologically active hCG using an expression system capable of producing large quantities of recombinant protein. Such a system can also serve as a model for investigating structure–function relationships and folding during subunit biosynthesis, in addition to providing sufficient quantities of hCG for immunocontraceptive studies.

Expression of hCG Using Pichia pastoris

Section I of the study deals with recombinant DNA expression of properly folded, biologically active hCG using the methylotrophic yeast Pichia pastoris. Section II focuses on expression and characterization of translationally fused and subunits using the same system.

Hyperexpression of glycoprotein hormones by recombinant DNA technology is challenging, as yields of properly folded and biologically active hormones are usually low regardless of the expression system. Glycosylation during biosynthesis is critical for correct folding of individual subunits. Another important feature is the presence of multiple disulfide linkages in both subunits, with three disulfide bridges in each subunit involved in forming the cystine knot structure. Correct disulfide bond pairing, as well as the order of disulfide bond formation, is essential for biological activity. Glycosylation of asparagine residues in the hCG subunit ensures correct disulfide bond pairing. Hence, glycosylation and disulfide bond formation are simultaneous and interrelated processes.

Earlier studies also implicate the redox environment within the endoplasmic reticulum and the involvement of Protein Disulfide Isomerase (PDI) and cell-specific chaperones in the folding and association of hCG subunits. These factors render in vitro folding of glycoprotein hormone subunits highly inefficient. This is a major limitation in bacterial expression systems, where hCG and subunits are not glycosylated and often aggregate into inclusion bodies, requiring denaturation and refolding. Mammalian systems can produce biologically active hCG but are limited by low yields, difficult scale-up, and purification challenges.

Advantages of Yeast Expression Systems

Yeast expression systems, such as Pichia pastoris, combine genetic manipulability and rapid growth with the eukaryotic machinery for post-translational modifications. Proteins passing through the secretory pathway undergo proteolytic maturation, glycosylation, and disulfide bond formation. Pichia has been used for high-level expression of many recombinant proteins. It allows easy scaling from shake flasks to high-density fermentors without loss of productivity, uses inexpensive defined media, and unlike Saccharomyces cerevisiae, does not overmannosylate proteins. The oligosaccharides produced by Pichia are of appropriate length (ManGlcNAc), avoiding problems of overglycosylation.

Expression of hCG Subunits

The subunit was expressed using the pPIC9K vector with a yeast mating factor signal peptide for secretion. A C-terminal hexahistidine tag facilitated Ni² affinity purification. Shake flask expression yielded ~24–27 mg/L of secreted subunit (phCG ). Immunological probes confirmed that the Pichia-expressed subunit was similar to native subunit and could anneal with native subunit to form a biologically active heterodimer.

The subunit was expressed using the same vector, yielding 2.7–3 mg/L of secreted protein (phCG ). Immunoassays confirmed similarity to the native subunit. Co-expression of and subunits in Pichia produced heterodimeric hCG (phCG) at 12–16 mg/L. Radioimmunoassays and monoclonal antibody binding confirmed immunological similarity to native hCG.

phCG could bind LH receptors with affinity comparable to native hCG and stimulated progesterone production in MA-10 Leydig cells. Maximal stimulation was twofold higher than native hCG, likely due to altered glycosylation.

Expression of Fusion Protein (hCG )

Single-chain fusion proteins, in which and subunits are genetically linked, overcome limitations of heterodimerization. In this study, the C-terminus of subunit was fused to the N-terminus of subunit without additional linker sequences (one glycine at the junction). The fusion protein (phCG ) maintained a conformation resembling native hCG, as confirmed by polyclonal and monoclonal antibodies.

Monoclonal antibody MAb 52/28, which recognizes an epitope formed only upon heterodimerization, confirmed formation of the receptor binding domain. phCG bound LH receptors with affinity similar to native hCG and was biologically active, with maximal stimulation equivalent to hCG. Compared to phCG, phCG was slightly less active.

Role of C-Terminal Subunit Residues

A mutant fusion protein (phCG ) lacked the five C-terminal amino acids of the subunit. Immunological assays showed overall conformation similar to phCG and native hCG, though subtle differences were noted in the subunit. MAb 52/28 confirmed formation of the heterodimer-specific epitope.

Receptor binding assays indicated phCG could bind LH receptors with similar affinity but produced lower maximal stimulation, acting as a partial agonist. Therefore, the five C-terminal residues are not essential for receptor binding but are critical for full signal transduction.

Conclusions

Pichia pastoris efficiently produces biologically active, immunologically similar hCG and fusion derivatives.

Fusion proteins allow structure–function studies without heterodimerization limitations.

Glycosylation, disulfide formation, and proper folding are essential for biological activity.

Deletion of C-terminal residues affects signal transduction but not receptor binding.

This system enables production of sufficient recombinant protein for detailed structural and functional analyses.

If you want, I can also create a version with all abbreviations spelled out and a glossary for hCG, PDI, RIA, etc., which is often useful for theses and publications