The flanking amino acids of the human follitropin beta-subunit 33-53 region are involved in assembly of the follitropin heterodimer.
Previous analyses of the topology of human follitropin (hFSH) with monoclonal antibodies and antipeptide antibodies have led to a current operating hypothesis that some amino acids within the hFSH beta 33-53 region are surface oriented, and others participate in subunit contact. Protein structural analysis predicts beta-turns within this region, and the immunochemical studies indicate that the ends may be involved in subunit contact. In this study, hFSH beta was mutagenized to change 34TRDL37 to 34AAAA37 or 48QKTCT52 to 48AAACA52, allowing us to study the ends of the hFSH beta 33-53 sequence contiguous with the hFSH beta sequence. Wild-type and mutant cDNAs were coexpressed with alpha-subunit cDNA in CHOPro-5 cells. Wild-type hFSH was secreted from cells cotransfected with wild-type hFSH alpha and hFSH beta cDNAs, as expected. However, heterodimeric hFSH was minimally detected in the medium from cells transfected with the 34TRDL37 mutant and was not detected in the case of the 48QKTCT52 mutant. Analysis of cell lysates (intracellular FSH) by immunoprecipitation and polyacrylamide gel electrophoresis showed that wild-type and mutant beta-subunits were indistinguishable and recoverable intact from each cell line. Additionally, analysis of lysates with a conformation-specific monoclonal antibody 3G3 revealed that similar levels of properly folded beta-subunit were produced in cells expressing wild-type or either mutated beta-subunit. These data indicate that the flanking amino acids of the hFSH beta 33-53 region, in particular 48QKTCT52, are critical for assembly of hFSH heterodimer.
School of Public Health, State University of New York, Albany 12201.
K E
KE
Roth
C
C
Liu
B A
BA
Shepard
J B
JB
Shaffer
J A
JA
Dias
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005640:Follicle Stimulating Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015345:Oligonucleotide Probes / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D005640:Follicle Stimulating Hormone