Isolation of a cDNA encoding a murine UDPgalactose:beta-D-galactosyl- 1,4-N-acetyl-D-glucosaminide alpha-1,3-galactosyltransferase: expression cloning by gene transfer.

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:beta-D-galactosyl-1,4-N-acetyl-D-glucosaminide alpha-1,3-galactosyltransferase [(alpha 1-3)GT; EC 2.4.1.151]. This library was transfected into COS-1 cells, which lack expression of (alpha 1-3)GT. Transfected cells containing functional (alpha 1-3)GT cDNAs were detected and isolated with a lectin that recognizes the surface-expressed glycoconjugate product of the (alpha 1-3)GT enzyme. One cloned (alpha 1-3)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(beta 1-4)GlcNAc to form Gal(alpha 1-3)Gal(beta 1-4)GlcNAc. This cloning approach may be generally applicable to the isolation of cDNAs encoding other mammalian glycosyltransferases.

A cloned human DNA restriction fragment determines expression of a GDP-L-fucose: beta-D-galactoside 2-alpha-L-fucosyltransferase in transfected cells. Evidence for isolation and transfer of the human H blood group locus.

We have described previously a gene transfer system for the isolation of human DNA sequences that determine expression of a mammalian GDP-fucose: beta-D-galactoside-2-alpha-L-fucosyltransferase (alpha-(1,2)-fucosyltransferase) (Ernst, L. K., Rajan, V. P., Larsen, R. D., Ruff, M. M., and Lowe, J. B. (1989) J. Biol. Chem. 264, 3436-3447). With this system, we found that de novo expression of the fucosyltransferase in mouse recipient cells was associated with the transfer and stable genomic integration of characteristic human DNA restriction fragments. We report here the results of experiments designed to determine the genetic origin of the fucosyltransferase determined by these sequences. First, we characterize the fucosyltransferases found in these mouse transfectants and in the human cell line used as a DNA donor. We compare their properties to those displayed by the human H and Secretor blood group fucosyltransferases. We find that the enzymes in the transfected cells have properties similar or identical to those of the human H alpha-(1,2)-fucosyltransferase. However, their properties differ significantly from the properties of the human Secretor alpha-(1,2)-fucosyltransferase and are also distinct from the properties of a murine alpha-(1,2)-fucosyltransferase. To confirm further that these transfected human sequences determine the H phenotype of the transfectants, we cloned the two human EcoRI restriction fragments common to each H-expressing secondary transfectant. The larger of these two fragments directs de novo expression of an alpha-(1,2)-fucosyltransferase when transfected into COS-1 cells. The pH activity profile of this alpha-(1,2)-fucosyltransferase and its apparent Michaelis constants for substrate and acceptor mirror those we determined for the human H alpha-(1,2)-fucosyltransferase. We conclude that genetic information sufficient to determine expression of this alpha-(1,2)-fucosyltransferase resides within the 3.4-kilobase pair human EcoRI restriction fragment and that this most likely represents the human H blood group locus.

Cholesterol flux between high density lipoproteins and cultured rat luteal cells.

Although high density lipoprotein (HDL) is a promoter of cholesterol efflux from peripheral cells, in steroidogenic tissues it has been shown to provide cholesterol for steroid synthesis. The present study examines the dynamics of cholesterol exchange between HDL particles and rat luteal cells. Cholesterol influx was measured by following the uptake of label from HDL particles labeled with [3H]cholesterol or [3H]cholesteryl linoleate. The efflux of cholesterol was simultaneously measured by incubating [3H]cholesterol-labeled luteal cells with unlabeled HDL. Conversion of endogenous and lipoprotein-derived steroids to progestins was also determined. The results showed that HDL promotes both influx and efflux of cholesterol in those cells. The amount of influx exceeds efflux, thus resulting in a net uptake of cholesterol from HDL by rat luteal cells. The relative utilization of endogenous vs. HDL-derived cholesterol for steroid synthesis was also examined. The results show that only a fraction of the HDL-derived cholesterol was converted directly to steroids. The fraction that was converted depended on the HDL concentration and had no apparent relation to the incubation time. These results show that although cholesterol flux in luteal cells is bidirectional in the presence of HDL, the influx exceeds the amount of efflux, and the internalized cholesterol is diluted with the endogenous cholesterol pool before it is converted to steroids.

Stable expression of blood group H determinants and GDP-L-fucose: beta-D-galactoside 2-alpha-L-fucosyltransferase in mouse cells after transfection with human DNA.

We report here the application of a genetic approach to identify and isolate human DNA sequences controlling the expression of a GDP-L-fucose: beta-D-galactoside 2-alpha-L-fucosyltransferase [alpha-1,2)fucosyltransferase). Mouse L cells were chosen as host cells for this scheme since they express the necessary substrate and acceptor molecules for surface display of blood group H Fuc alpha 1----2 G al linkages constructed by (alpha-1,2) fucosyltransferases. However, they do not express cell surface blood group H structures nor detectable (alpha-1,2)fucosyltransferase activity. We therefore asked if (alpha-1,2)fucosyltransferase activity could be expressed and detected in these cells after transfection with human DNA sequences. These cells were transfected with genomic DNA isolated from a human cell line (A431) that expresses (alpha-1,2)fucosyltransferase. A panning procedure and fluorescence-activated cell sorting were used to isolate a mouse transfectant cell line that expresses cell surface H Fuc alpha 1----2 Gal linkages and a cognate (alpha-1,2)fucosyltransferase. Southern blot analysis showed that the genome of this cell line contains several hundred kilobase pairs of human DNA. Genomic DNA from this primary transfectant was used to transfect mouse L cells, and several independent, H-expressing secondary transfectants were isolated by immunological selection. Each expresses an (alpha-1,2)fucosyltransferase. Southern blot analysis demonstrated that the genome of each secondary transfectant contains common, characteristic human DNA restriction fragments. These results show that transfected human DNA sequences determine expression of the (alpha-1,2)fucosyltransferases in the mouse transfectants, that these sequences represent a single locus, and that they are within or linked to specific human restriction fragments identifiable in each secondary transfectant. These sequences may represent a human (alpha-1,2)fucosyltransferase gene.

Differential uptake and metabolism of free and esterified cholesterol from high-density lipoproteins in the ovary.

Rat luteal cells utilize high-density lipoproteins (HDL) as a source of cholesterol for steroid synthesis. Both the free and esterified cholesterol of HDL are utilized by these cells. In this report, we have examined the relative uptake of free and esterified cholesterol of HDL by cultured rat luteal cells. Incubation of the cells with HDL labeled with [3H]cholesterol or [3H]cholesteryl linoleate resulted in 4-6-fold greater uptake of the free cholesterol compared to esterified cholesterol. The increased uptake of free cholesterol correlated with its utilization for progestin synthesis: utilization of HDL-derived free cholesterol was 3-6-fold higher than would be expected from its concentration in HDL. The differential uptake and utilization of free and esterified cholesterol was further examined using egg phosphatidylcholine liposomes containing cholesterol or cholesteryl linoleate as a probe. Liposomes containing free cholesterol were able to deliver cholesterol to luteal cells and support steroid synthesis in the absence of apolipoproteins, and the addition of apolipoprotein A-I (apo A-I) moderately increased the uptake and steroidogenesis. Similar experiments using cholesteryl linoleate/egg phosphatidylcholine liposomes showed that inclusion of apo A-I resulted in a pronounced increase in the uptake of cholesteryl linoleate and progestin synthesis. These experiments suggest that free cholesterol from HDL may be taken up by receptor-dependent and receptor-independent processes, whereas esterified cholesterol uptake requires a receptor-dependent process mediated by apolipoproteins.

Metabolism of high-density lipoproteins in cultured rat luteal cells.

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.

Interstitial cell heterogeneity in rat testes. I. Purification of collagenase-dispersed Leydig cells by unit gravity sedimentation and demonstration of binding sites for gonadotropin in light cells versus enhanced steroidogenesis in heavier cells.

Two testicular interstitial cell fractions, light and heavier, biochemically and morphologically distinct were obtained by a unit gravity sedimentation procedure. Binding sites for 125I-labeled human chorionic gonadotropin (hCG) were preferentially localized in the light cell fraction (apparent Kd = 2.02 X 10(-10) M; Bmax = 1.17 X 10(-5) nmol/2 X 10(6) cells). These cells did not synthesize testosterone in response to hCG, but the basal release of testosterone was higher than by cells in the heavier fraction (2.49 +/- 0.02 ng/2 X 10(6) cells in the light versus 0.22 +/- 0.00 ng/2 X 10(6) cells in the heavier fraction). The cells in the heavier fraction bound little or no hCG. The binding data from this fraction did not obey saturation kinetics, but testosterone levels were elevated 700-800% in the presence of hCG (i.e. basal value 0.22 +/- 0.00 ng/2 X 10(6) cells versus 1.81 +/- 0.04 ng/2 X 10(6) cells in hCG-stimulated cells). Electron microscopy revealed that heavier cells had features typical of Leydig cells such as large ovoid nucleus with peripherally located heterochromatin, numerous mitochondria with tubular cristae, some lipid droplets, extensively developed smooth endoplasmic reticulum, and well developed Golgi complex. The cells in the light fraction contained an ovoid nucleus with one or more deep infoldings, and their most notable cytoplasmic feature was the presence of numerous vacuoles of varying sizes and shapes. Based upon this and the investigation which follows (Bhalla, V.K., Flasch, M.V., Browne, E.S., Sohal, G.S., and Sharawy, M.M. (1987) J. Biol. Chem. 262, 5322-5332), we conclude that occupancy of high affinity hCG binding sites, generally assumed to be coupled to steroidogenesis, is not necessarily related to the elicitation of this biological response.

Involvement of microtubules in lipoprotein degradation and utilization for steroidogenesis in cultured rat luteal cells.

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.

Evidence that dissociation, not intracellular degradation, is the major pathway for removal of receptor-bound 125I-human chorionic gonadotropin in cultured rat luteal cells.

The nature of the labeled products released by cultured rat luteal cells pulse-labeled with 125I-human chorionic gonadotropin (hCG) was examined. After pulse labeling in a 3-h incubation, the cells containing receptor-bound 125I-hCG were incubated in fresh medium in the absence of 125I-hCG up to 48 h. The medium was collected at different time intervals and analyzed to determine the extent of degradation of 125I-hCG. The amounts of radioactivity remaining associated with the cells at these time intervals were also determined. Most of the released radioactivity could be precipitated with 10% trichloracetic acid and was identical in molecular weight to intact 125I-hCG as determined by gel filtration chromatography. After 20 h of reincubation, only less than 50% of the initially bound hormone remained on the cells. At this time point the cells were capable of rebinding 125I-hCG at levels comparable to the original when incubated with a fresh dose of the labeled hormone. The rebinding ability was not a result of de novo receptor synthesis since cycloheximide had no effect on this process. The results indicate that dissociation is the major pathway for release of hCG bound to cultured rat luteal cells and that receptors become functional again after dissociation of the hormone by a cycloheximide-independent process.