Molecular characterization and differential mRNA tissue distribution of rabbit apolipoprotein D.

Pr Provost,Pk Weech,Nm Tremblay,Yl Marcel,E Rassart

doi:10.1016/s0022-2275(20)42270-9

Abstract

We report for the first time the quantification of relative apolipoprotein D (apoD) mRNA concentrations in a wide selection of organs and a detailed characterization of the rabbit protein. ApoD cDNA clones were isolated from a rabbit testis cDNA library by screening with a human apoD cDNA-derived RNA probe. The 912 nucleotide sequence of rabbit apoD cDNA contains a unique reading frame coding for a protein sharing 80% homology with human apoD. The two sequences have two potential asparagine-linked glycosylation sites at the same positions, almost superimposable hydrophobicity plot, and the antigenic proteins show similar charge polymorphism, Mr, and lipoprotein distribution. This high degree of similarity shows that the rabbit system can be used as a model for apoD studies. Moreover, the two consensus sequences of the hydrophobic ligand carrier (alpha 2-microglobulin) family present in human apoD are also found in the rabbit protein and these sequences coincide with the most conserved regions. The distribution of apoD mRNA among rabbit organs was determined by Northern blot and quantitative dot blot analysis. The highest levels of mRNA were found in spleen, adrenal glands, lungs, brain, testis, and kidneys. Moderate or low concentrations were detected in all the other organs tested including liver and small intestine. Thus, our results show that the apoD gene is expressed mainly in peripheral organs, with levels as high as 59-fold that of the liver, unlike other apolipoproteins. We suggest that apoD exerts its main function locally in peripheral organs.

Highlights

We report for the first time the quantification of relative apolipoprotein D mRNA concentrations in a wide selection of organs and a detailed characterization of the rabbit protein
Monoclonal antibody anti-human apolipoprotein D (apoD) 4Ell reacted with human HDLonly, but monoclonal antibody 5G10 reacted with the reduced proteins of rabbit, pig, dog, cow, goat, and cynomolgusand rhesus monkeys (Fig. l), and sheep
1) The rabbit apoD protein cross reactswell withthe monoclonal antibody anti-human apoD 5G10 (Fig. 1)which reacts withan epitope composed of the polypeptide rather than the oligosaccharidesof apoD

Summary

Introduction

We report for the first time the quantification of relative apolipoprotein D (apoD) mRNA concentrations in a wide selection of organs and a detailed characterization of the rabbit protein. We have measured and compared the salient characteristics of rabbit and human apoD and its mRNA, and we report here for the first time a systematic and quantitative comparison of the apoD mRNA levels in organs This shows that the major concentrations were in spleen, adrenal gland, lung, brain, testis, and kidney, with only minor concentrationsin liver and intestine. Human apohpopmtein D (apoD)is a gtycop.oteinof apparent M,29,000 that was isolated and partially characterized by McConathy and Alaupovic [1,2], and subsequentlyby Drayna et al [3], Weech et al [4, 5], and Albers et al [6] In the plasma it is distributed mainly in the high density lipoproteins (HDL)with minor amountsin LDL, VLDL, and the plasma protein fraction (5 and references therein). It is associated with lecithin: cholesterol acyltransferase(LCAT) [6,7,8,9,10] and Abbmviations: apoD, apolipoprotein D; HDL, high density lipoproteins: LDL, low density lipoproteins: VLDL. wry low density lipoproteins: VHDL, very high density lipoproteins; LCXT, lecithin: cholesterol acyltransferase; RBP, retinol-bindingprotein; SDS, sodium dodecyl sulfate: PAGE, polyacrylamide gel electrophoresis: IEF, isoelectric focusing: SSC, sodium saline citrate; apoA-1, apolipoprotein A-I; bp, base pair: kbp. kilobase pair: apoE. apolipoprotein E

Methods

Results

Conclusion