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B A Shepard

Publications and source records attributed to B A Shepard.

5 recordsLinked to original sources
PubMed1993

Site-directed alanine mutagenesis of Phe33, Arg35, and Arg42-Ser43-Lys44 in the human gonadotropin alpha-subunit.

Residues Phe33 and Arg35, individually, and a composite mutation of residues Arg42, Ser43, and Lys44 were changed to alanine in the human glycoprotein hormone common alpha-subunit using site-directed mutagenesis. These specific residues are highly conserved across species and have by chemical modification and synthetic peptide approaches been implicated in the binding of human chorionic gonadotropin (hCG) to leutinizing hormone (LH) receptor. In the present study we tested the hypothesis that specific alpha-subunit amino acid residues which stabilize the hormone receptor interaction for hCG have the same function in human follicle-stimulating hormone (hFSH). Wild type or mutant alpha-subunit cDNAs were coexpressed with wild type hFSH or hCG beta cDNA in sialylation defective Chinese hamster ovary cells. Recombinant hormones were tested in a radioligand receptor competition assay, using rat testis membranes as a source of FSH and LH receptors. Mutant hFSH heterodimers F33A-FSH, R35A-FSH, Arg42-Ser43-Lys44/Ala42-Ala43-Ala44- FSH all displaced 125I-hFSH in a similar fashion, indicating that these residues are not important for binding of hFSH to the rat FSH receptor. On the other hand, F33A-CG evidenced a 5-fold decrease in binding, while R35A-CG had over a 100-fold decrease in binding to the rat LH receptor when compared to the wild type recombinant hCG. These data demonstrate that a receptor-binding site on the common alpha-subunit which is very important for hCG binding to LH receptor is not important for the binding of hFSH to FSH receptor. Our interpretation of these findings is that there are fundamental structural differences in the receptor interface contacts of the common alpha-subunit, which stabilize receptor binding among members of the glycoprotein hormone family.

Amino Acid Sequence↗
PubMed1993

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.

Amino Acid Sequence↗
PubMedDate not supplied

Pulmonary response to plasmid DNA and immunohistochemical localization of transgene expression.

Tracheal insufflation of chloramphenicol acetyltransferase (CAT) expression plasmid (pBL-CAT) DNA without any gene delivery system results in transfection of rat lungs (Am J Physiol. 1995;268:L1052-L1056). This study investigated the pulmonary response to tracheal insufflation of plasmid DNA and the immunohistochemical localization of transgene expression in the lungs. Insufflation of 300 micrograms pBL-CAT DNA resulted in lung transfection without causing pulmonary injury as judged from the protein content of alveolar lavage fluid, pleural effusion, and lung ultrastructure. There was, however, an acute alveolar inflammatory response at 6 h after insufflation due to contaminating endotoxin present in the plasmid DNA preparation. Reducing the amount of endotoxin from 0.022 to 0.0015 microgram per 300 micrograms pBL-CAT completely abolished the acute alveolar inflammatory response without affecting the lung transfecting efficiency of pBL-CAT. Immunohistochemistry revealed that insufflation of pBL-CAT transfected predominantly small airway epithelial cells.

Animals↗