Previous studies of human apo HDL using mass spectrometry have demonstrated that the molecular mass measurements could be compared with the calculated molecular weights of apoA-I (28078.6 Da) and apoA-II (17379.8 Da) (Pankhurst et al., 2003
). In this way, both the validation of the reported amino acid sequences, previously determined by protein analyses, as well as the detection of post-translational modifications of these apolipoproteins could be made. At the time our measurements were done, only partial amino acid sequences for these apolipoproteins existed for the chimpanzee (Scanu et al., 1974
); however, the entries in the genomic databases for the chimpanzee did provide us with complete sequences enabling such comparisons to be made for at least one of these great apes. The gorilla and the orangutan genomic entries appeared later. At first, we were wondering about the gorilla data because the molecular masses of both apoA-I and apoA-II were almost identical to reported human values (Pankhurst et al., 2003
). Also, the very low mass for orangutan apoA-II suggested that the protein might have undergone extensive enzymatic degradation. Nevertheless, later entries in the databases eliminated our concerns as discussed below. Unfortunately, there are no genomic entries for the bonobo.
Like humans, the genes for chimpanzee (NC_006478.2) and orangutan (ENSPPYG00000003904) apoA-I are located on chromosome 11. In both cases, the genes yield a 267 amino acid protein, consisting of an 18 amino acid signal sequence, a 6 amino acid propeptide and a 243 amino acid mature protein. Our molecular mass measurements for both the chimpanzee (28079.3 Da) and the gorilla (28078.7 Da) were very close to calculated molecular weight of human apoA-I. In agreement with these measurements, genomic data also have revealed that the primary sequences of the chimpanzee (NC_006478.2) and the gorilla (ENSGGOG00000008006) are identical to that of human apoA-I. The signal sequences and the propeptides of each of the great apes obtained from genomic data are also identical to the human sequences. However, the orangutan 243 amino acid sequence has five variations at residues 6, 53, 138, 167 and 197, with a threonine instead of a serine, a methionine instead of a valine, a histidine instead of a glutamine, threonine instead of a serine and a serine instead of a threonine, respectively (). As a result, orangutan apoA-I has a calculated molecular weight of 28133.7 Da that is in close agreement with one of the observed peaks with measured value of molecular mass 28134.0 Da. An examination of the entries in the Ensembl database for 15 different mammals indicates that this currently is the largest reported value for mammalian apoA-I. Our data for bonobo apoA-I were intermediate to the values of the orangutan and the other great apes, viz. 28108.8 Da. An observed post-translational modification of apoA-I due to loss of a C-terminal glutamine (Δ −128 Da) was observed in the bonobo and chimpanzee spectra, consistent with the observed average mass values of 27984.0 Da and 27953.3 Da, respectively.
Variations in great ape apoA-I
The gene for apoA-II is located on chromosome 1 in chimpanzees (ENSPTRG00000001580) and orangutans (FJ997614
), as is the case in humans. The respective genes encode an 18 amino acid signal peptide, a 5 amino acid propeptide and a 77 amino acid mature protein. Genomic data for human apoA-II (ENSP00000356969) reveal that the signal sequence and the propeptide are identical to those of the other great apes and that in each case a cysteine is present at residue 6. The mature apoA-II of the chimpanzee and the orangutan do vary from the human sequence as indicated in . Chimpanzee apoA-II has an aspartate and an asparagine at residues 8 and 9, respectively and a methionine at residue 68. The calculated molecular weight, after correcting for the conversion of the N-terminal glutamine to pyroglutamic acid, is 8735.9 Da. The resulting homodimer would then have a molecular weight of 17469.8 Da. Our measured molecular masses for the dimeric apoA-II of the chimpanzee (17469.7 Da) and the bonobo (17471.0 Da) were very close to this calculated value, suggesting that the sequences of these two great apes are probably the same. Our analyses of the orangutan apo HDL revealed that the apoA-II is currently the smallest to be reported. The three exons encoding this apolipoprotein initially were located in a 15 kb genomic entry (ABGA01295244.1
). In the third exon, the codon that would have encoded a glycine as the 71st residue instead has been replaced by a stop codon. Using the UCSC genome browser, these three exons also can be located between coordinates 90083319 and 90084578 on chromosome 1. The orangutan apoA-II also varies from the human sequence with a proline, a qlutamine, an asparagine and a lysine at residues 10, 37, 65 and 68, respectively (). The resulting 70 amino acid sequence has a corrected calculated molecular mass of 8031.3 Da. This would result in the dimer having a molecular weight of 16060.6 Da, in agreement with our measured molecular mass value. It should be noted that like the other great apes, most mammals studied also have a 77 amino acid apoA-II (Puppione et al., 2004
). Rats and mice apoA-II on the other hand are slightly larger with 79 amino acids (Puppione et al., 2004
). The sequence of gorilla apoA-II was obtained from a 5.4 kb entry (CABD01119658.1
) in the whole genome shotgun database. The resulting protein sequence, found to be identical to human apoA-II, yielded a corrected calculated molecular weight for the dimer of 17379.8 Da. Our average molecular mass value was 17380.7 Da.
Variations in great ape apoA-II
As was observed for apoA-I, there is also a truncated form of human apoA-II due to a loss of a C-terminal glutamine (Deterding et al., 2002
; Pankhurst et al., 2003
). This results in the formation of two dimers having lower molecular masses, viz.
a heterodimer and a homodimer. Both of these were detected in the apoA-II spectra of the chimpanzee. Lower molecular weight heterodimers were also present in the bonobo and gorilla spectra. Similar truncation also has been reported for monomeric mouse apoA-II, involving the loss of a C-terminal lysine (Puppione et al., 2006
). The apoA-II of great apes have a methionine residue at residue 26 and the chimpanzee apoA-II has an additional one at residue 68. Of the four groups of great apes studied here, only the orangutan apoA-II spectrum, with an additional peak at 16076.0 Da showed evidence of oxidation. The methionine at residue 68 in human apoA-II also is susceptible to oxidation and has been associated with the development of atherosclerosis (Pankhurst et al., 2003
Our studies of the HDL proteome of great apes have demonstrated that the similarities and differences in the apolipoprotein sequences determined from genomic data are consistent with our molecular mass measurements. A priori, one might assume that apolipoprotein molecular masses of the bonobo and the chimpanzee would be almost identical to their human counterparts, but only the chimpanzee apoA-I value is the same as humans and our molecular mass data indicate that both apoA-I and apoA-II of the bonobo are larger than the human apolipoproteins. On the other hand, the gorilla that diverged from the human-chimpanzee lineage between 7.7 and 9.4 million years ago has the same sequence for both apolipoproteins (Steiper et al., 2006). Interestingly, the orangutan, having the earlier time of divergence between 16.1 and 20.8 million years ago (Steiper et al., 2006), does not have the canonical 77 amino acid apoA-II.
As far as the involvement of the two apolipoproteins is concern, there is no reason to assume that their role will be different in great apes. However, the interaction of diet and metabolism represents potential sources for influencing evolutionary changes could give rise to different effects not seen in humans. The HDL are integrally involved in the metabolic process known as reverse cholesterol transport (Scanu and Edelstein, 2008). In this process the apoA-I containing HDL accept from extrahepatic tissue cholesterol which in turn is transesterified to cholesteryl ester through the action of lecithin cholesterol acyl transferase (LCAT) (Glomset, 1968
; Scanu and Edelstein, 2008). This enzyme is activated by apoA-I. The eventual delivery of cholesteryl esters to the liver involves the interaction of apoA-I with the scavenger receptor SR-B1, located in the plasma membrane of hepatocytes where the hydrolyzed cholesterol is converted to bile acids. The bulk of the released fatty acids in this reaction will be polyunsaturated, having been involved in a previous LCAT reaction (Glomset, 1968
). These, in turn, would be potential ligands for the peroxisome proliferator-activated receptors present in liver cells (Lee et al., 2003
). Although phylogenetically related, the diets of the other great apes nevertheless are different from humans. Being primarily herbivorous and frugiverous, one would expect that the livers of the other great apes would be metabolizing a higher ratio of omega-3 vs omega-6 fatty acids from the uptake of both HDL cholesteryl esters and TG-rich lipoproteins. As more information is obtained on the respective genomes of the great apes, future studies might explore how these and other metabolic processes are influenced by diet.