High-density lipoprotein (HDL) and (LDL)-density lipoprotein (LDL) are important lipoprotein fractions in human plasma. HDL is the most abundant lipoprotein particle and a negative risk factor of atherosclerosis while LDL is considered to possess atherogenic properties. The molecular mechanisms underlying the relationship between LDU/HDL and the development of atherosclerosis are not clear. Therefore, detailed information about the protein composition of LDU/HDL may contribute to reveal their role in atherogenesis and the mechanisms that lead to or protect from coronary disease in humans. Here, we sought to map the proteins in human LDL/HDL by a proteomic approach.
LDL and HDL were isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix assisted laser desorption/ionization-time of flight-mass spectrometry and with amino acid sequencing using electrospray ionization tandem mass spectrometry.
In LDL these procedures identified apo B-100, apo C-II, apo C-I (three isoforms), apo E (four isoforms), apo A-I (three isoforms), apo A-IV, apo J and apo M (three isoforms not previously described). In addition, three proteins that have not previously been identified in LDL were found: serum amyloid A-IV (two isoforms). calgranulin A and lysozyme C. The identities of apo M, calgranulin A, and lysozyme C were confirmed by sequence information obtained after collision-induced dissociation fragmentation of pep tides characteristic for these proteins. Moreover, the presence of lysozyme C was further corroborated by demonstrating enriched hydrolytic activity in LDL against Micrococcus lysodeikticus.
Identified proteins in HDL were: the dominating apo A-1 as seven isoforms, four of them with a modification pattern and one of them with retained propeptide, apo A-II, apo A-IV, apo C-I. apo C-II, apo C-III (two isoforms), apo E (five isoforms), apo J, the recently discovered apo M (two isoforms), serum amyloid A (two isoforms) and serum amyloid A-IV (six isoforms). Furthermore, alpha-1-antitrypsin was identified in HDL for the first time. Additionally, salivary alpha-amylase was identified as two isoforms in HDL2 , and apo L and aglycosylatcd apo A-II were identified in HDL3.
These results indicate that both LDL and HDL contains a number of apolipoproteins, many of them occurs in different isoforms. The demonstration, for the first time, that LDL contains calgranulin A, lysozyme C and apo J raises the possibility that LDL proteins may play hitherto unknown role(s) in immune and inflammatory reactions of the arterial wall. Additionally, new patterns of glycosylated apo A-I and apo A-II and new proteins; alpha-1-antitrypsin and salivary alpha-amylase were detected in HDL. Further investigations about these proteins may give new insight into the functional role of LDL and HDL in coronary artery disease.
2005. , 40 p.