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 horm one essential for human reproduction. Twenty hCG P-subunit residues, termed the seatbelt, are wrapped around alpha -subunit loop 2 (alpha2) and their positions "latched" by a disulfide formed by cysteines at the end of the se atbelt (Cys 110) and in the beta -subunit core (Cys 26). This unique arrang ement explains the stability of the heterodimer but raises questions as to how the two subunits combine. The seatbelt is latched in the free beta -sub unit, If the seatbelt remained latched during the process of subunit combin ation, formation of the heterodimer would require alpha2 and its attached o ligosaccharide to be threaded through a small beta -subunit hole. The subun its are known to combine during oxidizing conditions in vitro, and studies described here tested the idea that this requires transient disruption of t he latch disulfide, possibly as a consequence of the thioredoxin activity r eported in hCG. We observed that alkylating agents did not modify either cy steine in the latch disulfide (Cys 26 or Cys 110) during heterodimer format ion in several oxidizing conditions and had minimal influence on these cyst eines during combination in the presence of mild reductants (1-3 mM beta -m ercaptoethanol). Reducing agents appeared to accelerate subunit combination by disrupting a disulfide (Cys 93-Cys 100) that forms a loop within the se atbelt, thereby increasing the size of the beta -subunit hole. We propose a mechanism for hCG assembly in vitro that depends on movements of alpha2 an d the seatbelt and suggest that the process of glycoprotein hormone subunit combination may be useful for studying the movements of loops during prote in folding.