Apolipoprotein E synthesis in peripheral tissues of nonhuman primates.

D L Williams,P A Dawson,T C Newman,L L Rudel

doi:10.1016/s0021-9258(18)89574-6

Abstract

The tissue distribution of apolipoprotein (apo) E synthesis in the cynomolgus monkey, Macaca fascicularis, was determined via short-term organ culture with radiolabeled amino acid. Tissue extracts were reacted with antiserum to apo-E, and immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Newly synthesized apo-E was detected in liver, adrenal, testis, lung, spleen, mesenteric lymph node, and kidney. Peripheral and hepatic apo-E showed the same electrophoretic mobility. High resolution two-dimensional gel analysis showed that newly synthesized apo-E exists in two major isoforms in each tissue examined. Comparison of isoform patterns and mixing experiments showed that newly synthesized apo-E isoforms have identical charge properties in each tissue examined. These data indicate that numerous peripheral tissues synthesize apo-E that is indistinguishable from liver apo-E by the criteria tested. Measurements of relative synthetic capacities illustrate that apo-E is a moderately abundant protein product of a variety of peripheral tissues although quantitative differences in apo-E synthesis occur. Apo-E mRNA from cynomolgus monkey liver and human Hep G2 cells co-migrated with an electrophoretic mobility corresponding to approximately 1200 nucleotides. Apo-E mRNA from liver, brain, thymus, kidney, testis, lymph node, and spleen was the same size. Primer extension analysis yielded a cDNA product representing complete copying of the 5' untranslated region of human Hep G2 apo-E mRNA. A cDNA of identical size was produced with cynomolgus monkey apo-E mRNA from liver, spleen, brain, lymph node, kidney, lung, and thymus. These data suggest that transcription of the apo-E gene is initiated at or near the same site in each tissue examined.

Highlights

The tissue distributioonf apolipoprotein E syn- as well as HDL subfractions
Amino acid sequence of apo-E synthesis in peripheral tissues, we have extended our studies toa broad spectrum of organs froman experimental nonhuman primate model, the cynomolgus monkey
High resolution two dimensional gel analysis showed that the apo-E species made in peripheral tissues correspond in isoform pattern to plasma apo-E or newly synthesized hepatic apo-E

Summary

RESULTS

Detection of Apo-E Synthesis-To determine the tissue distribution of apo-E synthesis in the cynomolgus monkey, Macaca fascicularis, the experimental strategy was the same as used previously to examine apo-Esynthesisinhuman tissues [12] and apo-A1synthesis inperipheral chicken tissues [29]. After short-term organ culture with radiolabeled amino acids, extracts of various tissues were screened with antiserum againstapo-E, andthe immunoprecipitated proteins were analyzed by SDS-10% polyacrylamide gel electrophoresis. The final apo-E preparation migrated essentially as a single band with an apparenmt olecular weight of000 upon electrophoresis in SDS-12% polyacrylamide gels (Fig. 1, lane E ). The maximum amount of these bands observed in several preparations is shown in Fig. 1( laneE ) .The amino acid sequence of the N-terminal 20 residues showed the sequence to be identical to human apo-E [44]with the following substitutions: residue 5 (alanine), residue 8 (proline), residue 10 (lysine), residue 18 (alanine), residue 20 (glycine). The amino acid differences a t residues 5, 8, 18, and 20 could be due to single nucleotide changes from the sequence of human apo-E cDNA [41]

Spleen

E Synthesis

DISCUSSION