RESUMEN
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.
Asunto(s)
Apolipoproteínas/química , Lipoproteínas HDL/química , Secuencia de Aminoácidos , Animales , Apolipoproteínas/genética , Apolipoproteínas/aislamiento & purificación , Lipoproteínas HDL/genética , Lipoproteínas HDL/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Procesamiento Proteico-Postraduccional , Especificidad de la Especie , Espectrometría de Masa por Ionización de Electrospray , TripsinaRESUMEN
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.
Asunto(s)
Apolipoproteína A-II/análisis , Apolipoproteína A-II/química , Apolipoproteína A-I/análisis , Apolipoproteína A-I/química , Equidae/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Animales Domésticos , Apolipoproteína A-I/sangre , Apolipoproteína A-II/sangre , Cromatografía en Gel , Dimerización , Femenino , Caballos , Lipoproteínas HDL/sangre , Lipoproteínas HDL/química , Espectrometría de Masas , Peso MolecularRESUMEN
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.