Single chain glycoprotein hormones : structure-function studies

Abstract

Structure-Function Studies of Glycoprotein Hormones Using Fusion Protein Strategy

Abstract and Synopsis Introduction Structure-function studies of proteins aim to elucidate how individual amino acid residues are oriented to meet specific functional requirements. Advances such as recombinant DNA technology and site-directed mutagenesis have enabled selective manipulation of residues, greatly aiding structural chemists. These strategies are particularly useful in studying the complex glycoprotein hormone family, which exhibits diverse structural and functional features.

Expression System

The Pichia pastoris system was adopted for recombinant protein expression.

Recombinant hFSH and hTSH were expressed, with hFSH showing markedly higher yields than hTSH.

Proteins were purified using hydrophobic interaction chromatography, with hFSH purified to clinical-grade levels.

Fusion Protein Strategy

A novel derivative, single-chain hCG , was synthesized by fusing the -subunit C-terminus to the -subunit N-terminus.

Despite altered folding, the derivative retained receptor binding and biological activity.

Mutational analyses revealed:

Yields of single-chain proteins were lower than heterodimers.

Proline38 mutation in the -subunit, normally disruptive, was tolerated in the fusion protein, confirming the robustness of the tethering strategy.

Glycosylation at Asn52 was dispensable in the fusion protein, unlike in heterodimers.

Introduction of basic residues near the L1 loop produced superagonists with enhanced receptor affinity.

Functional Insights

Fusion of two -subunits (hCG ) yielded a receptor-binding but biologically inactive analog, acting as an antagonist of hormone action.

Structural analyses suggested local -subunit-like domains in fusion proteins, though insufficient for signal transduction.

These findings highlight the distinct roles of and subunits:

-subunit: receptor binding.

-subunit: signal transduction.

Conclusions

The fusion protein strategy successfully produced both agonists and antagonists of hormone action.

Mutational studies revealed critical domains for subunit interaction, receptor binding, and signal transduction.

This approach offers a powerful tool for engineering hormone analogs for therapeutic applications.

Future directions include crystallographic and NMR studies to resolve folding dynamics and receptor specificity.