No abstract

DegraWion of the peroxisomal enzymes fatty acyl-CoA oxidase and cut&se was studied in hepatocytes isolated from rats treated with clofibrate and from control rats. HepatocyLes were incubated in the absence of amino acids in order to ensure maximal flux through the autophagic pathway and in the presence of cycloheximidc to inhibit protein synthesis, (I) Degradation of the two psroxisomal enzymes in hepatocytes from clofibrate-fed rats, but not in hepatocytes from control rats, was much faster than that of other intracellular enzymes, This increased degradation of the pcroxisomal enzymes was almost completely prevented by 3-methyladeninr, an inhibitor of mscroautophagic squmtration. (2) The increased degradation of the peroxisomal enzymes was also inhibited by a long-chain (Cl6:O) and a very-longchain (C26:O) fatly acid, but not by Cl2:0, a medium-chain fatty acid, or by C&O, a short-chain fatty acid. These results provide direct evidence for the proposal thal autophagic sequestration can be highly selective [( 1987) Exp. Mol. Pathol. 46, 1 l4-1221. It is concluded that preferential aulophagy of peroxisomes is prevented when :hese organclles are supplied with their fatty acid substrates.

The peroxisomal oxidation of the long chain fatty acid palmitate (C16:0) and the very long chain fatty acids lignocerate (C24:0) and cerotate (C26:0) was studied in freshly prepared homogenates of cultured skin fibroblasts from control individuals and patients with peroxisomal disorders. The peroxisomal oxidation of the fatty acids is almost completely dependent on the addition of ATP, coenzyme A (CoA), Mg2+ and NAD'.However, the dependency of the oxidation of palmitate on the concentration of the cofactors differs markedly from that of the oxidation of lignocerate and cerotate.The peroxisomal oxidation of all three fatty acid substrates is markedly deficient in fibroblasts from patients with the Zellweger syndrome, the neonatal form of adrenoleukodystrophy and the infantile form of Refsum disease, in accordance with the deficiency of peroxisomes in these patients. In fibroblasts from patients with X-linked adrenoleukodystrophy the peroxisomal oxidation of lignocerate and cerotate is impaired, but not that of palmitate. Competition experiments indicate that in fibroblasts, as in rat liver, distinct enzyme systems are responsible for the oxidation of palmitate on the one hand and lignocerate and cerotate on the other hand. Fractionation studies indicate that in rat liver activation of cerotate and lignocerate to cerotoyl-CoA and lignoceroyl-CoA, respectively, occurs in two subcellular fractions, the endoplasmic reticulum and the peroxisomes but not in the mitochondria. In homogenates of fibroblasts from patients lacking peroxisomes there is a small (25%) but significant deficiency of the ability to activate very long chain fatty acids. This deficient activity of very long chain fatty acyl-CoA synthetase is also observed in fibroblast homogenates from patients with X-linked adrenoleukodystrophy. We conclude that X-linked adrenoleukodystrophy is caused by a deficiency of peroxisomal very long chain fatty acyl-CoA synthetase.

Pairs of cystine residues were introduced in the alpha- and beta-subunits of human choriogonadotropin at positions with optimal geometries for the formation of disulfide bonds. Using the homology with luteinizing hormone and follicle stimulating hormone, similar mutations were carried out in these glycoprotein hormones. In nearly all mutants the corresponding disulfide bonds were formed leading to a non-natural, covalent linkage between the alpha- and beta-subunits. The mutants typically display wild-type receptor binding and bioactivity. The mutants with non-natural intersubunit disulfide bonds display enhanced thermostabilities relative to the corresponding heterodimeric glycoprotein hormones, rendering them candidates for long acting gonadotropins with enhanced shelf lives.

Human choriogonadotropin (hCG) is a highly complex glycoprotein consisting of two non-covalently associated subunits. We aimed for the expression of a single-chain hCG in the soil amoebae Dictyostelium discoideum, a host which, in principle, provides simple genetics in combination with complex protein synthesis. To limit anticipated problems in mRNA translation, the first 30 bases of the coding sequence were altered to conform to the Dictyostelium preferred codon usage. We show that, immunologically, active single-chain hCG is indeed produced by Dictyostelium. Furthermore, this single-chain hCG is able to bind to the human luteinizing hormone/CG receptor and elicit a biological response. Its receptorbinding affinity is comparable to single-chain hCG produced by mammalian cells. We conclude that Dictyostelium is able to express bioactive highly complex heterologous glycoproteins.Keywords : gonadotropin; single chain; Dictyostelium; human choriogonadotropin; follitropin.The placental human choriogonadotropin (hCG) is involved in the maintenance of pregnancy in the early stages after conception and has important therapeutic applications. This gonadotropin belongs to the family of glycoprotein hormones, which also include follitropin, lutropin and thyrotropin. These hormones are heterodimeric proteins of around 30 kDa formed by a non-covalent association of a common A subunit and a hormone-specific β subunit. Both the A and β subunits of hCG contain two Nlinked oligosaccharide side chains that have an important impact on its conformation and biological activity. A unique feature of the hCG β-subunit is the carboxy-terminal peptide (CTP) which bears four serine-linked oligosaccharides. The major role of the glycosylated CTP seems to be the prolongation of the circulatory half-life of hCG [1].The biosynthesis of the glycoprotein hormones is a highly complex process. In the last decade, it has become clear that folding, assembly and secretion of gonadotropins is assisted by a large set of chaperones and folding enzymes, residing in the endoplasmic reticulum and the Golgi apparatus [2]. Since both the A and the β subunits contain a so-called cystine knot, it can be anticipated that protein disulphide isomerase plays a key role in the facilitation of the folding process [3]. In addition, it has been shown that the N-linked oligosaccharide side chains are required for proper folding, disulphide formation and secretion of hCG [4].The gonadotropins have been expressed in Chinese Hamster Ovary (CHO) cells, and their recombinant derivatives have bioCorrespondence to P. D. J.

The rhizomelic form of chondrodysplasia punctata (RCDP) is a peroxisomal disorder characterized biochemically by an impairment of plasmalogen biosynthesis and phytanate catabolism. We have now found that the maturation of peroxisomal 3-oxoacyl-CoA thiolase is impaired in fibroblasts from RCDP patients.To establish the subcellular localization of the 3-oxoacylCoA thiolase precursor protein, cultured skin fibroblasts were fractionated on a continuous Nycodenz gradient. Only a small amount of 3-oxoacyl-CoA thiolase activity was present in the catalase-containing (peroxisomal) fractions of RCDP fibroblasts in comparison with control fibroblasts. Moreover, the amount of thiolase protein in immunoblots of the catalase-containing fractions was below the limit of detection. Finally, the fl-oxidation of 1'4Cjpalmitoyl-CoA was found to be reduced in these fractions.We conclude that the mutation in RCDP leads to a partial deficiency of 3-oxoacyl-CoA thiolase activity in the peroxisomes and, concomitantly, an impairment in the ability to convert the precursor of this protein to the mature form. The reduction of 3-oxoacyl-CoA thiolase activity results in a decrease in the rate of peroxisomal f-oxidation of palmitoylCoA. However, the capacity of the peroxisomes to oxidize very-long-chain fatty acids must be sufficient to prevent excessive accumulation of these compounds in vivo. (J. Clin. Invest.

Several studies indicate that in human choriogonadotropin the N-linked oligosaccharide at position 52 of the A-subunit is important for bioactivity. We have generated choriogonadotropin mutants in which the A52 glycosylation site is removed and the A and β subunits are covalently linked by intersubunit disulfide bonds. These mutants display wild-type receptor binding and bioactivity. Furthermore, we show that removal of the A52 sugar leads to instability of heterodimeric choriogonadotropin. Therefore, we conclude that the A52 oligosaccharide of choriogonadotropin is not involved in signal transduction, but in the stability of the heterodimer.Keywords : human choriogonadotropin; bioactivity ; signal transduction ; protein engineering; glycoprotein.Human choriogonadotropin (CG), luteinizing hormone (LH) position plays a major role in signal transduction in CG [5] and FSH [6, 7]. and follicle-stimulating hormone (FSH) form the family of gonadotropins, which is involved in the regulation of the cellular Recently, we have shown that it is feasible to introduce covalent bonds between the A and β subunits of CG by introducing and endocrine function of the reproductive organs [1]. They consist of A and β subunits that are associated by hydrogen bonds intersubunit disulfide bonds [8]. This class of glycoprotein hormones displays wild-type receptor binding and biological activand hydrophobic contacts. In a given species the A subunit is identical in all glycoprotein hormones, while the β subunits are ity, whereas the thermostability of the hormones is enhanced considerably. Furthermore, these disulfide-bonded mutants were unique and confer receptor specificity on the hormones.A substantial part of the molecular mass of gonadotropins is designed to have overall conformations minimally disturbed relative to that of wild-type CG. Therefore, such molecules provide attributed to complex carbohydrates; in CG approximately 34% of the mass of the glycoprotein is due to complex carbohydrates. a very attractive system to study effects of mutations that in a heterodimeric context could interfere with the stability of the The β subunit of CG has four O-linked carbohydrates at Ser121, Ser127, Ser132 and Ser138. In addition, N-linked oligosaccha-heterodimer. In this paper we analyze the role of the AsnA52 glycosylation site in signal-transducing activities using this class rides are present at Asn52 and Asn78 on the A subunit and at Asn13 and Asn30 on the β subunit. Several studies have ad-of disulfide-bonded CG molecules. We will show that in stable CG, the A52 glycosylation site is not involved in signal transducdressed the role of the oligosaccharide chains on the gonadotropins [2]. It has been shown that the carbohydrate moieties are tion. involved in the folding and secretion of gonadotropins. Furthermore, glycosylation is essential for the plasma half-life of the glycoprotein hormones.MATERIALS AND METHODS The gonadotropins are amongst the most prominent examMaterials. Enzymes for preparing the expression vectors ples in glycobi...

No abstract