We have developed a genetic approach to isolate cloned cDNA sequences that determine expression of cell surface oligosaccharide structures and their cognate glycosyltransferases. A cDNA library was constructed in a mammalian expression vector by using mRNA from a murine cell line known to express a UDPgalactose:,B-D-galactosyl-1,4-N-acetyl-D-glucosaminide a-1,3-galactosyltransferase [(al-3)GT; EC 2.4.1.151]. This library was transfected into COS-1 cells, which lack expression of (al-3)GT. Transfected cells containing functional (al-3)GT cDNAs were detected and isolated with a lectin that recognizes the surface-expressed glycoconjugate product of the (al-3)GT enzyme. One cloned (al-3)GT cDNA was rescued from lectin-positive transfected cells. This cDNA contains a single long open reading frame that predicts a 394-amino-acid protein. No significant primary structure similarities were identified between this protein and other known sequences. However, the protein predicts a type II transmembrane topology similar to two other mammalian glycosyltransferases. This topology places the large COOH-terminal domain within the Golgi lumen; this domain was shown to be catalytically active when expressed in COS-1 cells as a portion of a secreted protein A fusion peptide. Biochemical analysis confirmed that this enzyme catalyzes a transglycosylation reaction between UDP-Gal and Gal(fi1-4)GlcNAc to form Gal(al-3)Gal(Ji1-4)GlcNAc. This cloning approach may be generally applicable to the isolation ofcDNAs encoding other mammalian glycosyltransferases.

11 beta-Hydroxysteroid dehydrogenase (11 beta-OHSD) catalyses the reversible conversion of corticosterone to inactive 11-dehydrocorticosterone, thus regulating glucocorticoid access to mineralocorticoid and perhaps glucocorticoid receptors in vivo. 11 beta-OHSD has been purified from rat liver and an encoding cDNA isolated from a liver library. However, several lines of indirect evidence suggest the existence of at least two isoforms of 11 beta-OHSD, one found predominantly in glucocorticoid receptor-rich tissues and the other restricted to aldosterone-selective mineralocorticoid target tissues and placenta. Here we have examined the effects of chronic (10 day) manipulations of sex-steroid levels on 11 beta-OHSD enzyme activity and mRNA expression in liver, kidney and hippocampus and present further evidence for the existence of a second 11 beta-OHSD isoform in kidney. Gonadectomized male and female rats were given testosterone, oestradiol or blank silicone elastomer capsules, controls were sham-operated. In male liver, gonadectomy+oestradiol treatment led to a dramatic decrease in both 11 beta-OHSD activity (69 +/- 8% decrease) and mRNA expression (97 +/- 1% decrease). Gonadectomy and testosterone replacement had no effect on male liver 11 beta-OHSD. However, in female liver, where 11 beta-OHSD activity is approximately 50% of that in male liver, gonadectomy resulted in a marked increase in 11 beta-OHSD activity (120 +/- 37% rise), which was reversed by oestradiol replacement but not testosterone treatment. In male kidney, gonadectomy+oestradiol treatment resulted in a marked increase in 11 beta-OHSD activity (103 +/- 4% rise). By contrast, 11 beta-OHSD mRNA expression was almost completely repressed (99 +/- 0.1% decrease) by oestradiol treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells isolated from superovulated rat ovaries metabolize low density lipoprotein (LDL) and high density lipoprotein (HDL) of human or rat origin and use the lipoprotein-derived cholesterol as a precursor for progesterone production. Under in vitro conditions, both lipoproteins are internalized and degraded in the lysosomes, although degradation of HDL is of lower magnitude than that of LDL. In this report we have examined the role of cellular microtubules in the internalization and degradation of human LDL and HDL in cultured rat luteal cells. The microtubule depolymerizing agents colchicine, podophyllotoxin, vinblastine, and nocodazole as well as taxol, deuterium oxide, and dimethyl sulfoxide, which are known to rapidly polymerize cellular tubulin into microtubules, were used to block the function of microtubules. When these antimicrotubule agents were included in the incubations, degradation of the apolipoproteins of [125I]iodo-LDL and [125I]iodo-HDL by the luteal cells was inhibited by 50-85% compared to untreated control values. Maximum inhibitory effects were observed when the cells were preincubated with the inhibitor for at least 4 h at 37 C before treatment with the labeled lipoprotein. Lipoprotein-stimulated progesterone production by luteal cells was also inhibited by 50% or more in the presence of antimicrotubule agents. However, basal and hCG-stimulated progesterone production were unaffected by these inhibitors. The binding of [125I]iodo-LDL and [125I]iodo-HDL to luteal cell plasma membrane receptors was not affected by the microtubule inhibitors. Although binding was unaffected and degradation was impaired in the presence of the inhibitors, there was no detectable accumulation of undegraded lipoprotein within the cells during the 24 h of study. From this study we conclude that the uptake and utilization of LDL and HDL by cultured rat luteal cells are mediated by cellular microtubules.

The uptake of cholesterol from high-density lipoproteins (HDL) labeled with 125I and [3H]cholesterol was examined in cultured rat luteal cells. Luteal cells were incubated with labeled HDL, following which the metabolic fate of the apolipoproteins and cholesterol moieties of the receptor-bound HDL were examined. About 50% of the originally bound HDL apolipoproteins were released into the medium in 24 h by a temperature-dependent process while only 5% of the HDL cholesterol was released unmetabolized. Inclusion of unlabeled HDL in the chase incubation resulted in increased release of apolipoprotein-derived radioactive products without significant change in the release of unmetabolized cholesterol. 60% of the apolipoprotein-derived radioactivity could be precipitated with trichloroacetic acid; the remaining trichloroacetic acid-soluble radioactive fraction was identified as [125I]iodotyrosine. Gel filtration chromatography of the chase-released material showed that the trichloroacetic acid-precipitable products, which contained no detectable amounts of cholesterol, eluted over a range of molecular sizes (9-80 kDa). No intact HDL was retroendocytosed. About 80% of trichloroacetic acid-precipitable products could be immunoadsorbed on anti-apolipoprotein A-I antibody immobilized on CNBr-activated Sepharose, suggesting the presence of fragments containing apolipoprotein A-I. This material was also capable of reassociating with native HDL. Lysosomal inhibitors were partially effective in inhibiting the amount of trichloroacetic acid-soluble products formed. The lysosomal degradation appeared to have no role in the uptake of HDL-derived cholesterol. These studies demonstrate preferential and total uptake of HDL cholesterol by luteal cells, with concomitant degradation of the lipoprotein.