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Lipoproteomics: mapping of proteins in LDL and HDL
Linköping University, Department of Molecular and Clinical Medicine, Occupational and Environmental Medicine. Linköping University, Faculty of Health Sciences.
2005 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
2005. , 40 p.
Series
Linköping Studies in Health Sciences. Thesis, ISSN 1100-6013 ; 73
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-31069Local ID: 16791ISBN: 91-8529-917-0 (print)OAI: oai:DiVA.org:liu-31069DiVA: diva2:251892
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2013-09-20
List of papers
1. Lipoproteomics I: Mapping of proteins in low-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry
Open this publication in new window or tab >>Lipoproteomics I: Mapping of proteins in low-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry
2005 (English)In: Proteomics, ISSN 1615-9853, Vol. 5, no 2, 551-565 p.Article in journal (Refereed) Published
Abstract [en]

The molecular mechanisms underlying the relationship between low-density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information about the protein composition of LDL may contribute to reveal its role in atherogenesis and the mechanisms that lead to coronary disease in humans. Here, we sought to map the proteins in human LDL by a proteomic approach. LDL was 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. These procedures identified apo B-100, apo C-II, apo C-III (three isoforms), apo E (four isoforms), apo A-I (two 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 peptides characteristic for these proteins. Moreover, the presence of lysozyme C was further corroborated by demonstrating enriched hydrolytic activity in LDL against Micrococcus lysodeikticus. These results indicate that in addition to the dominating apo B-100, LDL contains a number of other apolipoproteins, many of which occur in different isoforms. The demonstration, for the first time, that LDL contains calgranulin A and lysozyme C raises the possibility that LDL proteins may play hitherto unknown role(s) in immune and inflammatory reactions of the arterial wall.

Keyword
Low-density lipoprotein, Lysozyme, Matrix assisted laser desorption/ionization-time of flight-mass spectrometry, Tandem mass spectrometry, Two-dimensional gel electrophoresis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14350 (URN)10.1002/pmic.200300938 (DOI)
Available from: 2007-03-15 Created: 2007-03-15 Last updated: 2013-09-20
2. Lipoproteomics II: Mapping of proteins in high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry
Open this publication in new window or tab >>Lipoproteomics II: Mapping of proteins in high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry
2005 (English)In: Proteomics, ISSN 1615-9853, Vol. 5, no 5, 1431-1445 p.Article in journal (Refereed) Published
Abstract [en]

High-density lipoprotein (HDL) is the most abundant lipoprotein particle in the plasma and a negative risk factor of atherosclerosis. By using a proteomic approach it is possible to obtain detailed information about its protein content and protein modifications that may give new information about the physiological roles of HDL. In this study the two subfractions; HDL2 and HDL3, 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/ionisation time of flight mass spectrometry. Identified proteins in HDL were: the dominating apo A-I as six isoforms, four of them with a glycosylation pattern and one of them with retained propeptide, apolipoprotein (apo) A-II, apo A-IV, apo C-I, apo C-II, apo C-III (two isoforms), apo E (five isoforms), 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 a glycosylated apo A-II were identified in HDL3. Besides confirming the presence of different apolipoproteins, this study indicates new patterns of glycosylated apo A-I and apo A-II. Furthermore, the study reveals new proteins in HDL; alpha-1-antitrypsin and salivary alpha-amylase. Further investigations about these proteins may give new insight into the functional role of HDL in coronary artery diseases.

Keyword
High-density lipoprotein, Matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry, Two-dimensional gel electrophoresis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14351 (URN)10.1002/pmic.200401010 (DOI)
Available from: 2007-03-15 Created: 2007-03-15 Last updated: 2013-09-20

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