Molecular basis for “null” lipoprotein(a) phenotypes and the influence of apolipoprotein(a) size on plasma lipoprotein(a) level in the baboon.

A.L White,J.E Hixson,D.L Rainwater,R.E Lanford

doi:10.1016/s0021-9258(17)37076-x

Abstract

High plasma levels of lipoprotein(a) (Lp(a)) and its unique apolipoprotein, apo(a), are an independent risk factor for cardiovascular disease. Plasma Lp(a) levels vary over a 1000-fold range and are determined by the apo(a) locus, which has at least 34 alleles expressing apo(a) isoforms with molecular weights from < 300,000 to > 800,000. In addition, "null" apo(a) alleles produce no detectable plasma apo(a). We used primary cultures of baboon hepatocytes to investigate the molecular basis for null apo(a) phenotypes. Immunoprecipitation of apo(a) after radiolabeling of hepatocytes revealed that some null alleles gave rise to intracellular protein products that were not secreted. Pulse-chase analysis and endoglycosidase digests demonstrated that these proteins were retained in the endoplasmic reticulum. We also examined the molecular basis for the documented inverse correlation between apo(a) size and plasma Lp(a) concentration. Steady-state labeling and pulse-chase analysis of hepatocytes from animals expressing two isoforms of apo(a) revealed that the endoplasmic reticulum residence time of secreted apo(a) isoforms was determined by their size. This accounted for the inverse relationship between isoform size and level of secretion. We conclude that the efficiency of post-translational processing of apo(a) is a major determinant of plasma Lp(a) concentration.

Highlights

Highplasma levels of lipoprotein(a) (Lp(a))and its number of kringle 4 domains encoded [10,11,12], which varies unique apolipoprotein,apo(a),are an independent risk from approximately 12 to 51 [13]
Fornull apo(a) phenotypesI. mmunoprecipitation of The apo(a) locus contributes to 79of0t%he variation in plasma apo(a) after radiolabelingof hepatocytes revealed that Lp(a)levels[17].Apo(a)isoformsizeisinverselycorrelated some nullalleles gave rise to intracellular protein prod- with plasma Lp(a) concentration [7, 8, 12] and accounts for ucts that were not secreted
19-70% of the variation depending on the ethnic group [18]. Endoglycosidase digests demonstratedthat these pro- exceptions to the correlation between apo(a) size and teins were retained in the endoplasmic reticulum

Summary

EXPERIMENTAL PROCEDURES

NEN.Sheepanti-humanapoB polyclonal antibodywas from Boehringer Mannheim, and goat anti-human Lp(a) was from Biodesign, Kennebunkport, ME. To Golgi apparatus consistentwith it being a matureapo(a) determine whether the transcripptositive null allele gave rise polypeptide [28] These data confirm that the two smallest to an apo(a)protein product, cells were labeled to steady state intracellular forms of apo(a) represented the precursor and with ["%]methionine and ["S]cysteine, and apoB and apo(a) mature forms of the secreted K isoform [28].The largest intrawere immunoprecipitated from the cell lysate and cultureme- cellular form of apo(a) represented a product of the transcript dium and analyzed by SDS-PAGE (Fig. 2 A ). One apo(a) protein, which co-migrated with the middle the mRNA product from the transcript positive null allele in intracellular form, was recovered from the medium (Fig. 2 A ) . This was not due to a general defect in cell represented the precursor and matureforms of the secreted protein secretion since plasminogen

Cells Media

Findings

DISCUSSION