Joyce W. Lustbader

Mitochondrial Aβ: a potential focal point for neuronal metabolic dysfunction in Alzheimer’s disease

Although amyloid‐β peptide (Aβ) is the neurotoxic species implicated in the pathogenesis of Alzheimers disease (AD), mechanisms through which intracellular Aβ impairs cellular properties, resulting in neuronal dysfunction, remain to be clarified. Here we demonstrate that intracellular Aβ is present in mitochondria from brains of transgenic mice with targeted neuronal overexpression of mutant human amyloid precursor protein and AD patients. Aβ progressively accumulates in mitochondria and is associated with diminished enzymatic activity of respiratory chain complexes (III and IV) and a reduction in the rate of oxygen consumption. Importantly, mitochondria‐associated Aβ, principally Aβ42, was detected as early as 4 months, before extensive extracellular Aβ deposits. Our studies delineate a new means through which Aβ potentially impairs neuronal energetics, contributing to cellular dysfunction in AD.

Carbohydrate and peptide structure of the α- and β-subunits of human chorionic gonadotropin from normal and aberrant pregnancy and choriocarcinoma

Human chorionic gonadotropin (hCG), purified from the urine of 14 individuals with normal pregnancy, diabetic pregnancy, hydatidiform mole, or choriocarcinoma, plus two hCG standard preparations, was examined for concurrent peptide-sequence and asparagine (N)- and serine (O)-linked carbohydrate heterogeneity. Protein-sequence analysis was used to measure amino-terminal heterogeneity and the “nicking” of internal peptide bonds. The use of high-pH anion-exchange chromatography coupled with the increased sensitivity of pulsed amperometric detection (HPAE/PAD) revealed that distinct proportions of both hCG α- and β-subunits from normal and aberrant pregnancy are hyperglycosylated, and that it is the extent of the specific subunit hyperglycosylation that significantly increases in malignant disease.Peptide-bond nicking was restricted to a single linkage (β47–48) in normal and diabetic pregnancy, but occurred at two sites in standard preparations, at three sites in hydatidiform mole, and at three sites in choriocarcinoma β-subunit. In the carbohydrate moiety, α-subunit from normal pregnancy hCG contained non-fucosylated, mono-and biantennary N-linked structures (49.3 and 36.7%, means); fucosylated biantennary and triantennary oligosaccharides were also identified (7.3 and 6.9%). In choriocarcinoma α-subunit, the level of fucosylated biantennary increased, offset by a parallel decrease in the predominant biantennary structure of normal pregnancy (P<0.0001). The β-subunit from normal pregnancy hCG contained fucosylated and nonfucosylated biantennary N-linked structures; however, mono- and triantennary oligosaccharides were also identified (4.6 and 13.7%). For O-linked glycans, in β-subunit from normal pregnancy, disaccharidecore structure predominated, whereas tetrasaccharide-core structure was also detected (15.6%). A trend was demonstrated in β-subunit: the proportions of the nonpredominating N- and O-linked oligosaccharides increased stepwise from normal pregnancy to hydatidi-form mole to choriocarcinoma. The increases were: for monoantennary oligosaccharide, 4.6 to 6.8 to 11.2%; for triantennary, 13.7 to 26.7 to 51.5% and, for O-linked tetrasaccharide-core structure, 15.6 to 23.0 to 74.8%. For hCG from individual diabetic pregnancy, the principal N-linked structure (34.7%) was consistent with a biantennary oligosaccharide previously reported only in carcinoma; and sialylation of both N- and O-linked antennae was significantly decreased compared to that of normal pregnancy.Taken collectively, the distinctive patterns of subunit-specific, predominant oligosaccharides appear to reflect the steric effect of local protein structure during glycosylation processes. The evidence of alternative or “hyperbranched” glycoforms on both α- and β-subunits, seen at low levels in normal pregnancy and at increased or even predominant levels in malignant disease, suggests alternative substrate accessibility for Golgi processing enzymes, α1,6fucosyltransferase andN-acetylglucosaminyltransferase IV, in distinct proportions of subunit molecules.

Biomarkers of ovarian function in girls and women with classic galactosemia

OBJECTIVE To determine whether premature ovarian insufficiency (POI) associated with classic galactosemia results from a true impairment of ovarian function or from aberrant FSH. DESIGN Cross-sectional study. SETTING University research laboratory. PATIENT(S) Study subjects included 35 girls and women with galactosemia and 43 control girls and women between the ages of <1 and 51 years. INTERVENTION(S) Blood sampling and medical and reproductive histories were obtained. MAIN OUTCOME MEASUREMENT(S) We determined FSH and anti-Müllerian hormone (AMH) levels in subjects with and without classic galactosemia. FSH bioactivity was measured in a subset of girls and women with and without galactosemia who were not on hormone therapy. RESULT(S) FSH levels were significantly higher and AMH levels were significantly lower in our galactosemic cases relative to controls. FSH bioactivity did not significantly differ between cases and controls. CONCLUSION(S) Close to 90% of girls and women with classic galactosemia have a profound absence of ovarian function, a deficit that is evident shortly after birth, if not before. These patients have no evidence of abnormally functioning FSH. AMH levels can be assessed before menarche or after initiation of hormone therapy and may supplement FSH as a useful blood biomarker of ovarian function for patients with classic galactosemia.

Expression of human chorionic gonadotropin uniformly labeled with NMR isotpes in Chinese hamster ovary cells: An advance toward rapid determination of glycoprotein structures

SummaryMost secreted eukaryotic proteins are modified by glycosylation, and it has been difficult to solve their structures by crystallographic or NMR techniques because of problems posed by the presence of the carbohydrate. The structure of a chemically deglycosylated form of the human pregnancy hormone, human chorionic gonadotropin (hCG), has been solved by crystallographic methods. Since chemical deglycosylation may have induced changes in the structure, and since it is known that deglycosylated hCG is biologically inactive, the crystallographic structure requires confirmation by NMR techniques. Also, it has not been possible to determine the structures of the isolated subunits, nor the nature of interactions between the carbohydrate side chains and the protein back bone by crystallographic methods. Structural information via NMR techniques can be obtained from proteins in solution if they can be uniformly labeled with 13C and 15N isotopes. We report the first such uniform labeling of a glycoprotein using a universal 13C-and 15N-labeling medium to express 13C, 15N-labeled hCG, suitable for solving the structure in solution of the native, biologically active form of hCG as well as that of its free subunits. The 13C, 15N-labeled recombinant hCG and its separated subunits are shown to be nearly identical to urinary hCG reference preparations on the basis of protein chemical studies, immunochemistry, biological activity, and the capability of isolated hormone subunits to recombine to form biologically active hormone. Mass spectrometric analysis and preliminary NMR studies indicate that the isotopic labeling is uniform and greater than 90% after only two growth passages in the labeling media. One unexpected finding during subunit purification was that lyophilization of glycoproteins from trifluoroacetic acid HPLC buffers may result in the loss of a significant portion of sialic acid.

Efficacy of native and hyperglycosylated follicle-stimulating hormone analogs for promoting fertility in female mice.

OBJECTIVE To compare the efficacy of recombinant human FSH (rhFSH) with rhFSH with four additional O-linked carbohydrates (rhFSH-CTP), rhFSH with four additional N-linked carbohydrates (rhFSH-N4), and the current gold standard for rodent ovarian stimulation, pregnant mare serum gonadotropin (PMSG), on fertility parameters in mice. DESIGN Animal study. SETTING Academic research center. ANIMAL(S) Adult C57Bl/6J female mice. INTERVENTION(S) Ovarian stimulation with 5 IU of rhFSH, rhFSH-CTP, rhFSH-N4, or PMSG. Forty-six hours later, 5 IU of hCG was injected to promote ovulation and females were mated overnight. MAIN OUTCOME MEASURE(S) Eggs retrieved after ovulation, in vitro embryo development, delivery rate, and litter size. RESULT(S) The hyperglycosylated FSH analogs, rhFSH-CTP and rhFSH-N4, enhanced ovulation and embryo maturation significantly better than rhFSH. RhFSH-N4 produced more eggs (28.5 +/- 1.9 per mouse) and embryos (17.8 +/- 1.6) compared with rhFSH-CTP (18.3 +/- 1.2 and 9.0 +/- 1.0, respectively). Treatment with rhFSH, rhFSH-N4, and PMSG produced statistically equivalent delivery rates and litter sizes. The delivery rate was surprisingly lower with rhFSH-CTP (14%) compared with PMSG (33%). CONCLUSION(S) Compared with rhFSH, treatment with hyperglycosylated rhFSH-CTP and rhFSH-N4 led to superior rates of ovulated eggs and subsequent in vitro embryo development. RhFSH-N4 was equivalent to PMSG, while all of the fertility parameters studied were lower with rhFSH-CTP than with PMSG therapy.

High-level bacterial expression of a natively folded, soluble extracellular domain fusion protein of the human luteinizing hormone/chorionic gonadotropin receptor in the cytoplasm of Escherichia coli.

We have expressed the extracellular domain of the human luteinizing hormone/chorionic gonadotropin (hLH/CG) receptor as a fusion protein with thioredoxin in the cytoplasm of an Escherichia coli strain that contains mutations in both the thioredoxin reductase and glutathione reductase genes. The chimeric protein isolated following induction of expression is purified in a soluble form and binds hCG with an affinity approximating that of native receptor. This truncated form of the receptor displays the same specificity as intact hLH/CG receptor and does not bind human follicle stimulating hormone. This cytoplasmically produced protein is expressed at levels that exceed 10 mg/L. Expression of properly folded extracellular domain of the hLH/CG receptor in the cytoplasm of E. coli allows the facile and economic purification of large quantities of material. This will facilitate the determination of the structure of the hormone-binding domain of the glycoprotein receptor as well as the production of epitope-specific antibodies.

Expression and characterization of recombinant β-subunit hCG homodimer

We have linked two human chrionic gonadotropin (hCG) β-subunit cDNAs in tandem such that the expressed fusion protein consists of two mature β-subunits joined through the carboxy terminal peptide of the first β-subunit. A single glycine residue is inserted between the two subunits in the fusion protein. Chinese hamster ovary (CHO) cells transformed with a clone that contains the fused cDNAs express and secrete a protein that is consistent with it being a β-hCG homodimer protein. These β-homodimer molecules can recombine with two free α-subunits indicating that both β-subunits within the homodimer are likely folded in their native conformation. Our data also suggest that the two β-subunits fold upon each other as a globular protein and do not appear to exist as a simple fusion of two linear β-subunits. Further-more, the two β-monomer subunits in the fusion protein form a stable homodimer that can bind and activate the hLH/CG receptor specifically. Recombination of the fusion protein with α-subunits appears to favor an arrangement where two α-subunits combine with a single molecule of the fusion protein. The recombined molecule consists of four subunits and is comparable to two tethered hCG moieties, which constitutes a hCG dimer. This hormone dimer can bind and activate the hLH/CG receptor with an activity approximating that of native hCG.

Three-dimensional structures of gonadotropins

Most secreted proteins are modified post-translationally with the addition of carbohydrate. It has been difficult to use crystallography to solve the structures of these proteins due to the inherent heterogeneity of the carbohydrate. The structure of the chemically deglycosylated form (hydrogen fluoride treated) of human chorionic gonadotropin (hCG) has been solved through crystallographic techniques. Unfortunately this form of hCG is not biologically active, and exhibits immunochemical differences from native hormone. In addition, subunit interactions appear altered after chemical deglycosylation as indicated by the increased thermal stability of the HF-treated hormone. The Asn 52 glycan on the alpha-subunit of hCG has been identified as being required for biological activity, it is, therefore, of physiological importance to determine the structure of the hormone with its carbohydrate intact. Also, it has not been possible to obtain crystals of the individual glycosylated subunits of hCG. Therefore an alternative method to solve the structure of the biologically active form of the hormone in solution as well as its separated subunits is necessary. Structural information utilizing NMR techniques can be obtained from native hCG subunits in solution if they can be uniformly labeled with 13C and 15N isotopes. We have developed a universal nonradioactive isotope, labeling medium enriched in 13C and 15N which can be used to express uniformly labeled hCG from Chinese hamster ovary cells suitable for solving the structure of the individual subunits and ultimately that of the native, biologically active hormone. The isotopically labeled recombinant hCG and its purified subunits are essentially identical to urinary hCG on comparison by biochemical, immunochemical, biological activity and the ability of the isolated subunits to recombine to form a biologically active dimer. Mass spectrometric analysis and preliminary structural NMR data indicate that the labeling is uniform and there is greater than 90% incorporation, sufficient for complete structural determination studies. This labeled growth medium represents a technological advance which will enable the rapid solution of the structures of the other glycoprotein hormones, as well as other glycoproteins which have proven unsuitable for crystallographic study.

Development of a Fluorescence Based High Throughput Assay for Antagonists of the Human Chorionic Gonadotropin Receptor Extracellular Domain: Analysis of Peptide Inhibitors

A simple method for prompt fluorescent detection of inhibitors of human chorionic gonadotropin (hCG) binding to the extracellular domain of the human luteinizing hormone/chorionic gonadotropin (hLHICG) receptor was developed for high throughput screening (HTS). Construction and analysis of a recombinant phage that displays the extracellular binding domain of the hLH/CG receptor on its surface and specifically binds hCG was previously described. To facilitate the identification of molecules that disrupt the interaction of hCG with its receptor, a method for prompt fluorescent detection of these phage bound to hCG was developed. This technique is extremely sensitive and employs fluorescent labels (PBXL dyes) that are derived from red and blue-green algae. Antibodies labeled with PBXL dye were able to specifically detect phage that display the extracellular domain of the hLH/CG receptor when bound to hCG immobilized in 96-well microplates. Decreases in fluorescence correlate with the concentration of exogenous hCG or hCG antagonists in the assay. This prompt fluorescence detection assay was optimized in a 96-well format as a model system for HTS applications that target the receptors for the group of hormones known as the gonadotropins. Low-affinity molecules that disrupt binding of the phage-displayed receptor extracellular domain to hCG can be rapidly identified in this high throughput screen.

Human Chorionic Gonadotropin: Progress in Determining Its Tertiary Structure

In humans, maintenance of pregnancy requires extending the lifetime of the corpus luteum, which produces essential steroids. The signal for this maintenance is provided by human chorionic gonadotropin (hCG), a dimeric glycoprotein hormone produced by the trophoblast cells of the early embryo. The absence of hCG would result in rapid termination of early pregnancy.