Baboon apolipoprotein A-IV. Identification of Lys76-->Glu that distinguishes two common isoforms and detection of length polymorphisms at the carboxyl terminus.

Various protein isoforms have been identified for human apolipoprotein A-IV (apoA-IV). However, investigations of their physiological effects have been limited because of low frequencies for many of the apoA-IV variants. Recent discovery of extensive variation in baboon apoA-IV using isoelectric focusing (IEF) makes this primate species an excellent model for genetic studies of apoA-IV. In this study, the molecular basis for net charge differences between two common apoA-IV isoforms (I and E) was determined by cloning and sequencing of intestinal cDNAs from homozygous baboons. An A-->G substitution was found in the third amphipathic repeat of the E isoform. This substitution causes a Lys-->Glu substitution at amino acid position 76 (Lys76-->Glu), adding two negative charges to the E isoform compared to the I isoform, consistent with their relative mobilities on IEF gels. Restriction isotyping was used to identify the substitution in leukocyte DNA from 15 baboons that had been typed by IEF, thus verifying Lys76-->Glu as the basis for the charge differences between the I and E isoforms. Physiological effects of the Lys76-->Glu substitution on high density lipoprotein-C levels were investigated in 431 baboons carrying the E and I isoforms. These studies revealed that the I isoform was associated with higher levels of high density lipoprotein-C on a high cholesterol, saturated fat diet (p = 0.04). The cDNA sequences showed that the carboxyl terminus of baboon apoA-IV contains a region of hydrophilic repeats (Glu-Gln-X-Gln) that is the largest yet found in any species (nine repeats compared to three to five repeats in human, mouse, and rat). A common length polymorphism was identified that inserts a single amino acid to form a five amino acid repeat. This is the first report of this type of length variation (insertion of a single amino acid rather than insertion of an entire repeat) in this region. In addition, a rare variant was found that inserts an entire four-amino-acid repeat, similar to the human apoA-IV-0 isoform.

Baboon lecithin cholesterol acyltransferase (LCAT): cDNA sequences of two alleles, evolution, and gene expression.

Lecithin cholesterol acyltransferase (LCAT) is a key enzyme of cholesterol metabolism that catalyzes esterification of cholesterol for packaging in high-density lipoprotein (HDL) particles. In this study, we cloned and sequenced LCAT cDNA from baboon, a nonhuman primate model of atherosclerosis. LCAT sequences have been highly conserved over approximately 25 million years since the divergence of the baboon and human lineages. The baboon and human sequences are 97% identical at the nucleotide (nt) level and 98% identical at the amino acid (aa) level. Only 18% of the nt substitutions change the aa sequence (nonsynonymous substitutions). The substitutions between baboon and human LCAT do not alter key functional sites including the interfacial substrate active site, asparagine-linked glycosylation sites, or sites at which rare mutations cause human familial LCAT deficiencies. We also sequenced LCAT cDNA for a less common allele that is associated with higher LCAT activities and altered lipoprotein phenotypes. There were no sequence differences between the two alleles, which suggests that genotypic effects are most likely due to allelic differences in gene expression. The tissue specificity of LCAT expression was investigated using an RNase protection assay calibrated with known amounts of synthetic human LCAT RNA. In a survey of baboon tissues, the highest levels of LCAT mRNA were found in the cerebellum and liver and trace amounts in the ileum, spleen and cerebral cortex.

Alpha-myosin heavy chain cDNA structure and gene expression in adult, fetal, and premature baboon myocardium.

To examine cardiac myosin gene structure and expression in a non-human primate model for human heart development and disease, we have constructed a cDNA library from baboon atrium and used baboon beta-myosin heavy chain (beta-MHC)* cDNA probes to isolate atrial MHC clones. The nucleotide sequence of one such clone, lambda BMHC alpha 3, contains sequences that encode part of the light meromyosin region (LMM) and the 3' untranslated region of the baboon alpha-MHC. To study cardiac MHC gene transcription, we constructed probes from the baboon alpha-MHC cDNA for S1 nuclease analyses of RNA from atria and ventricles. To examine translational regulation of cardiac MHC gene expression, we used monoclonal antibodies (MAb) against specific alpha- and beta-MHC epitopes for Western blot analyses. In atria and ventricles from adult baboons, we detected predominantly alpha- and beta-MHC gene transcripts, respectively. In ventricles from fetal baboons at two stages of development (140 and 160 days gestation), we also detected predominantly beta-MHC gene transcripts and isoforms. To investigate changes induced by parturition, we obtained ventricles from baboons that were prematurely delivered at 140 days gestation and supported for 10 days in an extrauterine environment. In contrast to adult and fetal patterns, we observed an increase in alpha-MHC transcripts and isoforms in ventricles of premature baboons. Because alpha-MHC gene expression is increased in premature baboons (total age of 150 days) compared to their older 160 day fetal counterparts, the induction of ventricular alpha-MHC synthesis must have resulted from factor(s) associated with parturition or prolonged mechanical ventilation rather than at predetermined stages of gestational development.

Apolipoprotein(a) (Apo(a)) glycoprotein isoforms result from size differences in Apo(a) mRNA in baboons.

Like humans, baboons possess serum lipoprotein(a) that contains apolipoprotein(a) (apo(a], and baboon apo(a) also occurs in distinguishable glycoprotein isoforms. To investigate the molecular basis for isoform size variation, we isolated hepatic RNA from baboons possessing different apo(a) isoforms for Northern blot analyses with rhesus apo(a) cDNA probes. In a survey of 22 baboons, we detected a variety of sizes of apo(a) transcripts (5.2-11.2 kilobases) that corresponded with relative mobilities and numbers of serum apo(a) isoforms. In unrelated baboons possessing apo(a) isoforms of similar mobilities, we detected apo(a) transcripts with similar mobilities. In related baboons possessing apo(a) isoforms that were identical by descent, apo(a) transcripts were also identical in size. Using data from 22 baboons included in this study, we compared the sizes of 20 apo(a) isoforms and corresponding transcripts. Linear regression analysis established a highly significant correlation (p less than 0.001) between estimated apo(a) transcript size and glycoprotein mass (r2 = 0.996). From these results, we conclude that apo(a) glycoprotein isoforms are due to structural differences in apo(a) transcripts. However, we also noted exceptions to correspondence between apo(a) transcripts and isoforms that suggest the action of additional post-transcriptional control of serum apo(a) levels.

The baboon beta-myosin heavy-chain gene: construction and characterization of cDNA clones and gene expression in cardiac tissues.

We have constructed a cDNA library from baboon ventricle and have used a rabbit beta-myosin heavy chain (beta-MHC) cDNA probe to isolate cross-hybridizing clones. The nucleotide sequence of one such clone, lambda BMHC beta 14, contains a portion of the coding region of the light meromyosin (LMM) region and the 3'-untranslated region of the baboon ventricular MHC. This cDNA clone is identified as containing beta-MHC sequences on the basis of similarity with the 3'-untranslated regions of beta-MHC genes from man (96% homologous) and rat (71% homologous), and dissimilarity with the 3'-untranslated region of the rat alpha-MHC gene (25% homologous). Alignment and comparison of the baboon cDNA nucleotide sequence with a human cDNA sequence reveal two amino acid substitutions in the LMM region that cause differences in their hydrophilicity profiles. These differences may alter MHC functions such as filament assembly. We have used baboon beta-MHC cDNA clones to construct probes for S1 nuclease protection studies to detect and to distinguish cardiac MHC gene transcripts in baboon ventricle, atrium, and diaphragm. As in human tissues, beta-MHC gene transcripts are detected in RNA from baboon ventricle and diaphragm. In baboon atrium, we detect beta-MHC gene transcripts as well as transcripts that may represent expression of the alpha-MHC gene. This study represents the first examination of cardiac MHC genes and gene expression in tissues from a large mammal that is closely related to man.