Jacqueline Etienne

Comparison of the cDNA and amino acid sequences of lipoprotein lipase in eight species

By aligning nucleotide and amino acid sequences of lipoprotein lipase in eight species (man, pig, cow, sheep, mouse, rat, guinea-pig and chicken), we found that the main domains (catalytic, N-glycosylation and putative heparin binding sites) are well conserved. The longest identical amino acid chain was encoded by a sequence between the end of exon 2 and the beginning of exon 3, emphasizing the importance of this region which encodes the beta 5-loop of the active site, among other domains. Exon 10 is entirely untranslated in the seven mammals studied here and contains species-characteristic deletions, insertions or elements rich in A or A + T. In chicken, the beginning of exon 10 is translated. These eight previously unreported alignments could be a useful tool for further studies on LPL function.

Interferon-gamma inhibits lipoprotein lipase in human monocyte-derived macrophages

Lipoprotein lipase (LPL) (EC 3.1.1.34) hydrolyzes triacylglycerols of very low density lipoproteins and chylomicrons. It is produced by several cell types, including macrophages, which are frequent in atherosclerotic lesions. The atherosclerotic plaque also contains activated T lymphocytes. We therefore investigated the possible regulatory effect of the T lymphocyte-derived lymphokine interferon-gamma (IFN-gamma) on macrophage LPL. Human monocyte-derived macrophages were treated with recombinant IFN-gamma or conditioned medium from activated peripheral blood mononuclear cells for 3 days. LPL activity was thereafter measured in the culture medium and in cell homogenates. The enzyme protein was detected at a cellular level by immunocytochemistry and immunopredicipitation. Recombinant IFN-gamma caused a profound decrease in macrophage LPL secretion. The IFN-gamma-treated cells, however, still contained immunodetectable enzyme and the decrease in secretion was apparently only partly due to an inhibited synthesis. Conditioned medium from activated peripheral blood mononuclear cells also drastically decreased the macrophage LPL secretion. When the conditioned medium was treated with antibodies against IFN-gamma, its down-regulating effect on macrophage LPL was totally removed. The data indicate that IFN-gamma is inhibiting macrophage LPL at least in part via a reduction of LPL synthesis. A local release of IFN-gamma may be important in the pathogenesis of atherosclerosis by affecting the lipid accumulation in the lesion.

Sequence of rat lipoprotein lipase-encoding cDNA

A rat lipoprotein lipase (LPL)-encoding cDNA (LPL) has been entirely sequenced and compared to the sequences of all the LPL cDNAs reported in other species. As expected, high homology was found between the coding exons. The putative catalytic triad, Ser132, Asp156, His241, according to human numbering, is conserved in rat. As is the case in mouse, an Asn444 present in human LPL is also missing. The major divergences between human, mouse and rat LPLs were observed in the untranslated exon 10, where (i) the rat cDNA exhibits a 157-bp insertion and an 81-bp deletion relative to human; (ii) neither the B1 repeat nor the homopurine stretch reported in mouse can be recognized, and (iii) the rat cDNA displays several A+T-rich stretches.

Maturation and secretion of lipoprotein lipase in cultured adipose cells. II. Effects of tunicamycin on activation and secretion of the enzyme

The effects of N-linked glycosylation on the activation and secretion of lipoprotein lipase were studied in Ob17 cells. The cells were first depleted of any activity and enzyme content by cycloheximide treatment and of precursors of oligosaccharide chains by tunicamycin. The repletion of lipoprotein lipase content was studied in these cells maintained in the presence of tunicamycin after cycloheximide removal. During the repletion phase, the EC50 values of inhibition by tunicamycin (approx. 0.2 microgram/ml) of the incorporation of labeled glucose, mannose or galactose into trichloroacetic acid-insoluble material were found to be identical. Under these conditions, the rate of protein synthesis was maximally decreased by 30%. The results showed clearly that the recovery in lipoprotein lipase activity was parallel to the recovery in hexose incorporation, no activity being recovered in the absence of glycosylation. An inactive form of lipoprotein lipase from tunicamycin-treated cells was detected by competition experiments with mature active lipoprotein lipase for the binding to immobilized antilipoprotein lipase antibodies, as well as by immunofluorescence staining. SDS-polyacrylamide gel electrophoresis and Western blots of cellular extracts and of extracellular media, obtained after tunicamycin-treated cells were exposed to heparin, revealed a single immunodetectable Mr 52 000 protein, whereas a single Mr 57 000 protein was detected in control cells. Therefore, the results indicate that the acquisition by lipoprotein lipase of a catalytically active conformation is linked directly or indirectly to glycosylation. Despite this lack of activation, the lipoprotein lipase molecule was able to migrate intracellularily and to undergo secretion after heparin stimulation of the tunicamycin-treated cells.

Regional adipocyte precursors in the female rat. Influence of ovarian factors.

A flow cytometric immunofluorescence procedure utilizing a specific antibody to rat adipose tissue lipoprotein lipase (LPL) was developed to quantify differentiated and undifferentiated preadipocytes present in the adipose tissue vascular stroma. This method is highly sensitive and specific for cells capable of synthesizing LPL in significant quantities. Pubescence in female rats was associated with an increase in differentiated preadipocytes and in fat cell number with enlargement of the fat depots in the perirenal, parametrial, and the subcutaneous dorsal and femoral regions. A concomitant decline in the percentage of undifferentiated preadipocytes occurred in all but the femoral depot. Ovariectomy reduced pubertal adipose growth in the femoral and parametrial but not the dorsal or perirenal regions. Furthermore, the femoral undifferentiated preadipocyte pool was not preserved in the ovariectomized animals. Thus, ovarian factors influence the pubescence-associated regional preadipocyte differentiation and conversion to adipocytes. The femoral depot contains an ovarian-dependent infinite pool of fat cell precursors. These features could account for the association between ovarian hormones and body fat topography.

Antibody against rat adipose tissue lipoprotein lipase

To facilitate detailed studies of rat adipose tissue lipoprotein lipase regulation, a high titre polyclonal antibody was raised against purified rat adipose tissue lipoprotein lipase (in a goat). The first stage of the purification of the lipoprotein lipase was carried out with heparin-Sepharose affinity chromatography. In the second stage we took advantage of the binding property of lipoprotein lipase to ampholytes. These ampholytes, used during this second step, do not have to be eliminated prior to injecting the enzyme preparation into the animal. They have neither toxic nor antigenic effects on the animal; moreover, their presence does not affect the antigenic potency of the lipoprotein lipase. When pre-incubated with a constant amount of adipose tissue lipoprotein lipase (8 mU/75 microliter), an equal volume of the antiserum raised either pure or diluted up to 1/50 resulted in complete inhibition of enzyme activity, and half maximal inhibition was observed at a dilution of 1/800. The antibody was effective in inhibiting rat heart lipoprotein lipase but not salt-resistant hepatic lipase. Immunodiffusion revealed a single line of precipitation between this antibody and the adipose tissue lipoprotein lipase.

Intracellular localization of lipoprotein lipase in adipose cells

Subcellular localization of lipoprotein lipase has been examined in differentiated Ob17 adipose cells. No patent activity is detectable in carefully homogenized cells. All latent activity can be unmasked by disrupting membrane structures with neutral detergents. The sequestration of lipoprotein lipase in closed membrane structures is supported by experiments of immunotitration with anti-lipoprotein lipase antibodies and by experiments showing a full protection of the masked activity against proteolytic attack by trypsin. The intracellular distribution of lipoprotein lipase investigated by immunofluorescence staining and by isopycnic centrifugation indicates that a large proportion of the enzyme is located in the Golgi apparatus, in which the activation of the enzyme is likely to take place (C. Vannier et al. (1985) J. Biol. Chem. 260, 4424-4431). Altogether, the results are in favor of a localization of lipoprotein lipase in adipose cells as being typical of that of a secretory protein and underline the absence of lipoprotein lipase in the cell cytoplasm.

Post-heparin lipolytic activity with no hepatic triacylglycerol lipase involved in a mammalian species.

It was found that lipolytic activity in bovine post-heparin plasma differed from that of other mammalian species by the fact that intravenous heparin induced the release of lipoprotein lipase but not hepatic triacylglycerol lipase. Initially, this fact was strongly suspected when no remaining lipolytic activity could be found after whole bovine post-heparin plasma had been tested with either 1 M NaCl or antiserum against lipoprotein lipase. This was further confirmed by using heparin-Sepharose affinity chromatography when the entire lipolytic activity was eluted with 1.5 M NaCl but none with 0.4 or 0.7 M NaCl. The active fraction had lipoprotein lipase characteristics, i.e. it required serum activators to produce optimum activity and was fully inhibited by NaCl of high molarity and by anti-lipoprotein lipase antiserum. Neither the different doses of heparin nor the various times of sampling altered the results. This raises the question whether hepatic triacylglycerol lipase is absent from the bovine liver or whether this enzyme is present but cannot be released by heparin.

L'activité phospholipasique A2 des plaquettes de rat

Summary Rat blood platelets show phospholipase A2 activities twenty times higher than human platelets. The breakdown of PE at pH 7.2 is linear for 15 minutes. There is no degradation at pH

Activité phospholipasique A2 du sérum de rat: Association de deux protéines

Abstract In the rat, blood coagulation is accompanied by activation of a plasma phospholipase A2 due to a factor contained in blood platelets and released in plasma by platelet lysis. This activity can be obtained in vitro by treating plasma devoid of platelets with a platelet lysate. The results reported in this communication show the activity of serum phospholipase to result from an association of two proteins, inactive when isolated, one originating from plasma, the other from platelets, which can be broken down by fractionation on a column of Blue Sepharose CL-6B.