Biomedical subjects

A K Miller-Lindholm

Publications and source records attributed to A K Miller-Lindholm.

4 recordsLinked to original sources

PubMed2000

Functional contributions of noncysteine residues within the cystine knots of human chorionic gonadotropin subunits.

Human chorionic gonadotropin (hCG) is a heterodimeric member of a family of cystine knot-containing proteins that contain the consensus sequences Cys-X(1)-Gly-X(2)-Cys and Cys-X(3)-Cys. Previously, we characterized the contributions that cystine residues of the hCG subunit cystine knots make in folding, assembly, and bioactivity. Here, we determined the contributions that noncysteine residues make in hCG folding, secretion, and assembly. When the X(1), X(2), and X(3) residues of hCG-alpha and -beta were substituted by swapping their respective cystine knot motifs, the resulting chimeras appeared to fold correctly and were efficiently secreted. However, assembly of the chimeras with their wild type partner was almost completely abrogated. No single amino acid substitution completely accounted for the assembly inhibition, although the X(2) residue made the greatest individual contribution. Analysis by tryptic mapping, high performance liquid chromatography, and SDS-polyacrylamide gel electrophoresis revealed that substitution of the central Gly in the Cys-X(1)-Gly-X(2)-Cys sequence of either the alpha- or beta-subunit cystine knot resulted in non-native disulfide bond formation and subunit misfolding. This occurred even when the most conservative change possible (Gly --> Ala) was made. From these studies we conclude that all three "X" residues within the hCG cystine knots are collectively, but not individually, required for the formation of assembly-competent hCG subunits and that the invariant Gly residue is required for efficient cystine knot formation and subunit folding.

Chorionic Gonadotropin↗

PubMed2000

Determination of the DNA sequences of acetylcholinesterase and butyrylcholinesterase from cat and demonstration of the existence of both in cat plasma.

Cat serum contains 0.5 mg/L of butyrylcholinesterase (BChE, EC 3.1.1. 8) and 0.3 mg/L of acetylcholinesterase (AChE, EC 3.1.1.7); this can be compared with 5 mg/mL and < 0.01 mg/L, respectively, in human serum. Cat BChE differed from human BChE in the steady-state turnover of butyrylthiocholine, having a 3-fold higher k(cat) and 2-fold higher K(m) and K(ss) values. Sequencing of the cat BCHE cDNA revealed 70 amino acid differences between cat and human BChE, three of which could account for these kinetic differences. These amino acids, which were located in the region of the active site, were Phe398Ile, Pro285Leu, and Ala277Leu (where the first amino acid was found in human and the second in cat). Sequencing genomic DNA for cat and human ACHE demonstrated that there were 33 amino acid differences between the cat and human AChE enzymes, but that there were no differences in the active site region. In addition, a polymorphism in intron 3 of the human ACHE gene was detected, as well as a silent polymorphism at Y116 of the cat ACHE gene.

Acetylcholinesterase↗

PubMed1999

A naturally occurring genetic variant in the human chorionic gonadotropin-beta gene 5 is assembly inefficient.

The hCGbeta gene family is composed of six homologous genes linked in tandem repeat on chromosome 19; the order of the genes is 7, 8, 5, 1, 2, and 3. Previous studies have shown that hCGbeta gene 5 is highly expressed during the first trimester of pregnancy. The purpose of our study was to identify naturally occurring polymorphisms in hCGbeta gene 5 and determine whether these alterations affected hCG function. The data presented here show that hCGbeta gene 5 was highly conserved in the 334 asymptomatic individuals and 41 infertile patients examined for polymorphisms using PCR followed by single stranded conformational polymorphism analysis. Most of the polymorphisms detected were either silent or located in intron regions. However, one genetic variant identified in beta gene 5 exon 3 was a G to A transition that changed the naturally occurring valine residue to methionine in codon 79 (V79M) in 4.2% of the random population studied. The V79M polymorphism was always linked to a silent C to T transition in codon 82 (tyrosine). To determine whether betaV79M hCG had biological properties that differed from those of wild-type hCG, a beta-subunit containing the V79M substitution was created by site-directed mutagenesis and was coexpressed with the glycoprotein hormone alpha-subunit in Chinese hamster ovary cells and 293T cells. When we examined betaV79M hCG biosynthesis, we detected atypical betaV79M hCG folding intermediates, including a betaV79M conformational variant that resulted in a beta-subunit with impaired ability to assemble with the alpha-subunit. The inefficient assembly of betaV79M hCG appeared to be independent of beta-subunit glycosylation or of the cell type studied, but, rather, was due to the inability of the betaV79M subunit to fold correctly. The majority of the V79M beta-subunit synthesized was secreted as unassembled free beta. Although the amount of alphabeta hCG heterodimer formed and secreted by betaV79M-producing cells was less than that by wild-type beta-producing cells, the hCG that was secreted as alphabeta V79M heterodimer exhibited biological activity indistinguishable from that of wild-type hCG.

Abortion, Spontaneous↗

PubMed1997

Human chorionic gonadotropin-beta gene expression in first trimester placenta.

The hCGbeta gene family contains six genes linked in tandem on chromosome 19 and labeled beta genes 7, 8, 5, 1, 2, and 3. Previous studies on a small number of placentas have indicated that beta gene 5 was the most highly expressed gene during the first trimester of pregnancy, followed by genes 3 and 8. Beta genes 7, 1, and 2 were expressed at very low levels. The purpose of this study was to determine 1) whether this pattern of expression was typical during normal pregnancy by sampling a large number of first trimester placentas, and 2) whether there was a correlation between gestational age and the pattern of hCGbeta gene expression. Total RNA from 27 first trimester placentas varying in age from 6-16 weeks was reverse transcribed into complementary DNA. The complementary DNA was amplified by PCR, and the amount of DNA representative of each hCGbeta gene was quantified by Genescan analysis. In 14 of the 27 placentas, hCGbeta gene 5 accounted for 50% or more of the total beta messenger RNA expressed. Beta gene 3 was expressed at levels ranging from 1-42% of the total, and beta gene 8 expression ranged from 12-32% of the total. Gene 7 expression was less than 3% of the total beta expression in all 27 placentas. Although there appeared to be a trend toward lower expression of beta gene 3 in placentas beyond 10 weeks gestational age, there was no correlation of the pattern of beta expression with placental age. Beta gene expression was also examined in two blighted ova, a spontaneous abortion sample, and a hydatidiform mole as well as in cultured JAR choriocarcinoma cells. With the exception of JAR cells, these abnormal tissues had low levels of gene 3 expression, but these levels were within the range of the patterns observed in normal placentas. These data suggest that it is the total amount of hCGbeta gene expression rather than the expression of individual beta genes that is important for the maintenance of normal pregnancy.

Abortion, Spontaneous↗