Higher primates, but not New World monkeys, have a duplicate set of enhancers flanking their apoC-I genes.

Previous studies have demonstrated that the apoC-I gene and its pseudogene on human chromosome 19 are flanked by a duplicate set of enhancers. Multienhancers, ME.1 and ME.2, are located upstream from the genes and the hepatic control region enhancers, HCR.1 and HCR.2, are located downstream. The duplication of the enhancers has been thought to have occurred when the apoC-I gene was duplicated during primate evolution. Currently, the only primate data are for the human enhancers. Examining the genome of other primates (great and lesser apes, Old and New World monkeys), it was possible to locate the duplicate set of enhancers in apes and Old World monkeys. However, only a single set was found in New World monkeys. These observations provide additional evidence that the apoC-I gene and the flanking enhancers underwent duplication after the divergence of Old and New World monkeys.

Mass spectral measurements of the apoHDL in horse (Equus caballus) cerebrospinal fluid.

As a continuation of our proteogenomic studies of equine apolipoproteins, we have obtained molecular masses for several of the apolipoproteins associated with the HDL in horse cerebrospinal fluid (CSF). Using electrospray-ionization mass spectrometry (ESI-MS), we report on values for apolipoproteins, A-I and A-II, as well as acylated apoA-I. In comparison with our previously published data on equine plasma apolipoproteins, there appears to be a higher percentage of acylated apoA-I in the CSF than in plasma. As was the case in plasma, apoA-II circulates as a homodimer. These studies also revealed a protein with a mass of 34,468Da that we are speculating is the value for horse apoE.

Homocysteinylated albumin promotes increased monocyte-endothelial cell adhesion and up-regulation of MCP1, Hsp60 and ADAM17.

RATIONALE: The cardiovascular risk factor homocysteine is mainly bound to proteins in human plasma, and it has been hypothesized that homocysteinylated proteins are important mediators of the toxic effects of hyperhomocysteinemia. It has been recently demonstrated that homocysteinylated proteins are elevated in hemodialysis patients, a high cardiovascular risk population, and that homocysteinylated albumin shows altered properties. OBJECTIVE: Aim of this work was to investigate the effects of homocysteinylated albumin - the circulating form of this amino acid, utilized at the concentration present in uremia - on monocyte adhesion to a human endothelial cell culture monolayer and the relevant molecular changes induced at both cell levels. METHODS AND RESULTS: Treated endothelial cells showed a significant increase in monocyte adhesion. Endothelial cells showed after treatment a significant, specific and time-dependent increase in ICAM1 and VCAM1. Expression profiling and real time PCR, as well as protein analysis, showed an increase in the expression of genes encoding for chemokines/cytokines regulating the adhesion process and mediators of vascular remodeling (ADAM17, MCP1, and Hsp60). The mature form of ADAM17 was also increased as well as Tnf-α released in the cell medium. At monocyte level, treatment induced up-regulation of ICAM1, MCP1 and its receptor CCR2. CONCLUSIONS: Treatment with homocysteinylated albumin specifically increases monocyte adhesion to endothelial cells through up-regulation of effectors involved in vascular remodeling.

Mass spectrometric measurements of the apolipoproteins of bovine (Bos taurus) HDL.

As is the case in most mammals, high density lipoproteins (HDL) also comprise the major group of lipid carriers that circulate in bovine (Bos taurus) blood. As a continuation of our proteogenomic studies of mammalian apolipoproteins, we have obtained molecular masses for several of the apolipoproteins associated with bovine HDL. The major apolipoprotein on the HDL surface is apoA-I, but other apolipoproteins were also detected. Using electrospray-ionization mass spectrometry (ESI-MS), we report on values for apolipoproteins, A-I, proA-I and A-II, as well as post-translationally modified apoA-I. Analyses of tryptic fragments did reveal the presence of apoA-IV and apoC-III. However, in contrast to our previous studies of other mammalian HDL, we did not detect apoC-I. Interestingly, examination of the current assembly for the bovine genome does not show any evidence for an apoC-I gene.

Detection of two distinct forms of apoC-I in great apes.

ApoC-I, the smallest of the soluble apolipoproteins, associates with both TG-rich lipoproteins and HDL. Mass spectral analyses of human apoC-I previously had demonstrated that in the circulation there are two forms, either a 57 amino acid protein or a 55 amino acid protein, due to the loss of two amino acids from the N-terminus. In our analyses of the apolipoproteins of the other great apes by mass spectrometry, four forms of apoC-I were detected. Two of these showed a high degree of identity to the mature and truncated forms of human apoC-I. The other two were homologous to the virtual protein and its truncated form that are encoded by a human pseudogene. In humans, the genes for apoC-I and its pseudogene are located on chromosome 19, the pseudogene being 2.5 kb downstream from the apoC-I gene. Based on the similarity between the apoC-I gene and the pseudogene, it has been concluded that the latter arose from the former as a result of gene duplication approximately 35 million years ago. Interestingly, the virtual protein encoded by the pseudogene is acidic, not basic like apoC-I. In the chimpanzee, there also are two genes for apoC-I, the one upstream encodes a basic protein and the downstream gene, rather than being a pseudogene, encodes an acidic protein (P86336). In addition to reporting on the molecular masses of great ape apoC-I, we were able to clearly demonstrate by "Top-down" sequencing that the acidic form arose from a separate gene. In our analyses, we have measured the molecular masses of apoC-I associated with the HDL of the following great apes: bonobo (Pan paniscus), chimpanzee (Pan troglodytes), and the Sumatran orangutan (Pongo abelii). Genomic variations in chromosome 19 among great apes, baboons and macaques as they relate to both genes for apoC-I and the pseudogene are compared and discussed.

Mass spectral analyses of the two major apolipoproteins of great ape high density lipoproteins.

The two major apolipoproteins associated with human and chimpanzee (Pan troglodytes) high density lipoproteins (HDL) are apoA-I and dimeric apoA-II. Although humans are closely related to great apes, apolipoprotein data do not exist for bonobos (Pan paniscus), western lowland gorillas (Gorilla gorilla gorilla) and the Sumatran orangutans (Pongo abelii). In the absence of any data, other great apes simply have been assumed to have dimeric apoA-II while other primates and most other mammals have been shown to have monomeric apoA-II. Using mass spectrometry, we have measured the molecular masses of apoA-I and apoA-II associated with the HDL of these great apes. Each was observed to have dimeric apoA-II. Being phylogenetically related, one would anticipate these apolipoproteins having a high percentage of invariant sequences when compared with human apolipoproteins. However, the orangutan, which diverged from the human lineage between 16 and 21 million years ago, had an apoA-II with the lowest monomeric mass, 8031.3 Da and the highest apoA-I value, 28,311.7 Da, currently reported for various mammals. Interestingly, the gorilla that diverged from the lineage leading to the human­chimpanzee branch after the orangutan had almost identical mass values to those reported for human apoA-I and apoA-II. But chimpanzee and the bonobo that diverged more recently had identical apoA-II mass values that were slightly larger than reported for the human apolipoprotein. The chimpanzee A-I mass values were very close to those of humans; however, the bonobo had values intermediate to the molecular masses of orangutan and the other great apes. With the already existing genomic data for chimpanzee and the recent entries for the orangutan and gorilla, we were able to demonstrate a close agreement between our mass spectral data and the calculated molecular weights determined from the predicted primary sequences of the respective apolipoproteins. Post-translational modification of these apolipoproteins, involving truncation and oxidation of methionine, are also reported.

Mass spectral analysis of the apolipoproteins on dog (Canis lupus familiaris) high density lipoproteins. Detection of apolipoprotein A-II.

In a recent study, we reported the detection of apoA-II associated with the plasma high density lipoproteins of pigs that were previously thought to lack or to have this apolipoprotein in trace amounts. Dogs have also been reported to lack this apolipoprotein; however, genomic data have revealed that the gene for apoA-II is present on chromosome 38. Prompted by this finding, we have carried out detailed mass spectral measurements on dog apo HDL. The molecular mass of apoA-II was obtained as well as values for proapoA-I, apoA-I, apoC-I. In each case, the measured values were found to be in excellent agreement with the corresponding molecular weights calculated from genomic data. Following reverse-phase chromatography, tryptic fragments in selected fractions were analyzed by tandem mass spectrometry (MSMS). In addition to apoA-I, proapoA-I and apoA-II, enzymatic fragments from both apoC-II and apoA-IV were detected. Post-translational modification (PTM) of apoA-I, involving glycosylation, oxidation of a single methionine and acylation, were also noted. We also report on the sequencing of apoC-I using "Top Down" mass spectrometry analysis.

Mass spectral analysis of the apolipoproteins on mouse high density lipoproteins. Detection of post-translational modifications.

Using mass spectrometry, we have recently reported on molecular masses of the apolipoproteins associated with porcine and equine HDL. In addition to obtaining accurate masses for the various apolipoproteins, we also were able to detect mass variations due to post-translational modifications. In the present study, we have used these same approaches to characterize the apolipoproteins in two inbred mouse strains, C57BL/6 and BALB/c. Comparing our molecular mass data with calculated values for molecular weight, we were able to identify the correct sequences for several of the major apolipoproteins. Analyses were carried out on the apolipoproteins of ultracentrifugally isolated HDL. Prior to analyses by electrospray ionization mass spectrometry (ESI-MS), the apolipoproteins were separated either by size exclusion or reverse phase chromatography. The molecular masses of apoA-I, proapoA-I, apoA-II, proapoA-II, apoC-I and apoC-III were obtained. Comparing the values obtained for the two strains, differences in the molecular masses of apoA-I, apoA-II and apoC-III were observed. In this study, post-translationally modified apolipoproteins, involving loss of amino acids from both the N- and C-termini, oxidation of methionine residues and possible acylation, were noted following reverse-phase separation. Further analyses by tandem mass spectrometry (MSMS) done on the tryptic digests of apolipoproteins separated by reverse phase chromatography enabled us to confirm sequence differences between the two strains, to verify selected apoA-I sequences that had been entered into the GenBank and to identify which methionines in apoA-I, apoC-III and apoE had been converted to methionine sulfoxides.

Mass spectral analysis of domestic and wild equine apoA-I and A-II: detection of unique dimeric forms of apoA-II.

In pigs, humans, chimpanzees and probably other great apes, a cysteine at residue 6 enables apolipoprotein A-II to form a homodimer. However, the apoA-IIs of other primates, lacking a cysteine residue, are monomeric. We have already reported that horse apoA-IIs form homodimers due also to a cysteine at residue 6. In this study, we wanted to determine whether other equine apoA-IIs might be monomeric. The high density lipoproteins were ultracentrifugally isolated from the plasmas of a horse (Equus caballus), a donkey (Equus asinus) and five wild equines: two types of zebras (Equus zebra hartmannae and Equus zebra quagga boehmi), a Przewalski's horse (Equus przewalskii), a Somali ass (Equus africanus somalicus) and a kiang (Equus kiang holdereri). Using liquid chromatography with electrospray-ionization mass spectrometry, we were able to obtain accurate values for the molecular masses of apoA-I and apoA-II. Homodimeric apoA-IIs were observed in each of the animals studied. The donkey had unique dimers, consisting of the proapolipoprotein A-II linked by a disulfide bond either to a mature apoA-II monomer or another proapoA-II. In addition, our data indicate that small amounts of apoA-I and apoA-II apparently are acylated.

Mass spectral analysis of pig (Sus scrofa) apo HDL: Identification of pig apoA-II, a dimeric apolipoprotein.

Comparative studies of mammalian high density lipoproteins have clearly indicated that the major apolipoprotein is apoA-I and in some mammals apoA-II is the second major apolipoprotein. However, in pigs, apoA-II has been considered to be either present in trace amounts or absent. Recently, cDNA sequences for pigs A-II have been entered into the database. Translation of these sequences revealed that pig A-II consisted of 77 amino acids and that a cysteine residue was at residue 6. The A-II of three other mammals, chimpanzees, horses and humans, also has a cysteine residue at this position. As a result of a disulfide bond formed between monomers, the A-II in each of these cases circulates as a homodimer. Using electrospray-ionization mass spectrometry (ESI-MS), we obtained molecular mass data demonstrating that dimeric apoA-II is also present in pig plasma. In addition to being the first to report on the presence of apoA-II in pig plasma, we also obtained values for the molecular masses of apoA-I, apoC-III, apoD and serum amyloid A protein.

Sequence of horse (Equus caballus) apoA-II. Another example of a dimer forming apolipoprotein.

Apolipoprotein A-II, the second major apolipoprotein of human HDL, also has been observed in a variety of mammals; however, it is either present in trace amounts or absent in other mammals. In humans and chimpanzee, and probably in other great apes, apoA-II with a cysteine at residue 6 is able to form a homodimer. In other primates as well as other mammals, apoA-II, lacking a cysteine residue, is monomeric. However, horse HDL has been reported to contain dimeric apoA-II that following reduction forms monomers. In this report, we extend these observations by reporting on the first complete sequence for a horse apolipoprotein and by demonstrating that horse apoA-II also contains a cysteine residue at position 6. Both the intact protein and its enzymatic fragments were analyzed by chemical sequence analysis and time-of-flight MALDI-MS (matrix assisted laser desorption ionization mass spectrometry). We also obtained molecular mass data on dimeric and monomeric apoA-II using electrospray-ionization mass spectrometry (ESI-MS). The data are compared with other mammalian sequences of apoA-II and are discussed in terms of resulting similarities and variations in the primary sequences.

Characterization of lipoproteins in cerebrospinal fluid of mares during pregnancy and lactation.

OBJECTIVE: To measure apolipoproteins in cerebrospinal fluid (CSF) from healthy mares and to determine whether CSF concentrations of apolipoproteins change during pregnancy and lactation. ANIMALS: 5 healthy pregnant mares. PROCEDURE: 2 sets of CSF samples were obtained; initial samples were obtained 10 to 30 days before parturition (mean, 18 days; median, 17 days), and second samples were obtained 19 to 26 days after parturition (mean, 23 days; median, 23 days). Cerebrospinal fluid was collected from the lumbosacral subarachnoid space of standing horses by use of routine collection techniques. Cerebrospinal fluid cholesterol concentrations were measured by use of a sensitive enzymatic assay. Ultracentrifugal fractions of CSF lipoproteins were characterized by determining the distribution of apolipoproteins, using polyacrylamide gel electrophoresis. RESULTS: Analyses of isolated ultracentrifugal fractions by polyacrylamide gel electrophoresis revealed 2 apolipoproteins, with the expected molecular weights for apolipoprotein E and apolipoprotein A-I. No significant differences were observed between pre- and postpartum values in mares. The concentration of cholesterol in CSF fluid of mares was comparable to values reported in other mammals. CONCLUSIONS AND CLINICAL RELEVANCE: Apolipoprotein E in CSF of horses is a major apolipoprotein associated with high-density lipoproteins, which is similar to findings in other mammals. Additional characterization of the role of apolipoproteins in mammalian CSF may provide critical insight into various degenerative neurologic disease processes.