LETTERS TO THE EDITOR N-Glycosylation of apolipoprotein A1 in cardiovascular diseases Dear Editor, A very interesting paper by Cubedo et al1 has been recently published ‘‘Glycoproteome of human Apo A-I: N- and O-glycosylated forms are increased in acute myocardial infarction patients,’’ which significantly contributes to our field in the clinical application of proteomics in cardiovascular diseases. The authors demonstrated (among other results) that both the N- and O-glycosylation of apolipoprotein A1 (apoA1) changed in patient plasma during the first 3 days after an acute myocardial infarction (AMI) event. In our recent study,2 we observed high molecular weight forms of apoA1 (hm-apoA1) of approximately 50 kDa in the plasma samples of patients with AMI, unstable angina pectoris (UAP), and stable angina pectoris (SAP). These hm-apoA1 were detected using 1- and 2-dimensional Western blot analysis and seemed to occur at different levels among the studied cohorts. The apoA1 N-glycosylated forms described by Cubedo et al in AMI patients seem to correspond to those observed in our published work; therefore, we decided to verify this assumption and to estimate their presence in other cardiovascular diseases, SAP, and UAP, in addition to AMI. Pooled plasma samples (n 5 10 for each cohort) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by Western blotting; apoA1 bands were visualized using an anti-apoA1 antibody conjugated with peroxidase (ab20784; Abcam, Cambridge, UK), a chemiluminescent substrate, and a G-box iChemi XT4 digital imaging device (Syngene Europe, Cambridge, UK). All procedures have been previously described in detail.2,3 A deglycosylation assay was performed according to Zielinska et al4 using
Reprint requests: Pavel Majek, Institute of Hematology and Blood Transfusion, U Nemocnice 1, 128 20 Prague 2, Czech Republic; e-mail: [email protected] Submitted for publication August 7, 2014; accepted for publication September 6, 2014. Translational Research 2015;165:360–362. 1931-5244/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2014.09.003
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Fig 1. Western blot analysis. Pooled plasma samples from patients with SAP, UAP, and AMI were used together with control samples (C) for a deglycosylation assay (1 or 2 indicate the addition of PNGase F). Lane 10 illustrates sodium dodecyl sulfate–polyacrylamide gel electrophoresis (nonreducing conditions) of the isolated apoA1. Asterisks indicate bands containing apoA1, as identified by mass spectrometry. apoA1, apolipoprotein A1; AMI, acute myocardial infarction; SAP, stable angina pectoris; UAP, unstable angina pectoris; Mw, molecular weight; PNGase F, peptide-N-glycosidase F.
peptide-N-glycosidase F (G1549; Sigma-Aldrich, St. Louis, Missouri). The results are illustrated in Fig 1. It is obvious that hm-apoA1 were detected in the UAP and AMI groups (lanes 6 and 8), weakly detected in SAP (lane 4), and almost not detected in the control group (lane 2), which is in accordance with our previous observation. Herein, we show that after deglycosylation, 2 additional lowered molecular weight bands were detected (50 and 35 kDa); these bands were observed in all the groups with almost equal intensities (lanes 3, 5, 7, and 9). These results show that the hm-apoA1 are N-glycosylated and, in particular, are presented in all the studied groups as estimated by deglycosylation. Moreover, the results show that the N-glycosylation pattern of hm-apoA1 (the degree and position of N-glycosylation) is very likely altered among the groups. The presence of glycosylation restricts the interaction of the antibody with the hm-apoA1, which results in limited detection (lanes 2 and 4), yet the bands can still be detected after deglycosylation (lanes 3 and 5). To highlight the changes observed and to make the results
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Fig 2. Comparison of band intensities. The band intensities were compared before and after deglycosylation (the positions of the bands are indicated in (C) using ImageJ software (A). A relative comparison of the same bands is illustrated in (B). AMI, acute myocardial infarction; SAP, stable angina pectoris; UAP, unstable angina pectoris; PNGase F, peptide-N-glycosidase F.
comparable, our data were further processed with ImageJ software.5 Fig 2, A shows the relative comparison of band intensities before and after deglycosylation (intensities were normalized to the total apoA1 level). For better clarity, the same bands are presented normalized with respect to the control group (Fig 2, B). The differences in intensity of hm-apoA1 among the studied groups are obvious when compared with the deglycosylated hm-apoA1. To eliminate the possibility of a nonspecific antibody interaction (false detection), apoA1 and hm-apoA1 were isolated using a Dynabeads Protein G immunoprecipitation kit (Invitrogen, Carlsbad, California) according to the manufacturer’s instructions from pooled UAP and AMI samples, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (nonreducing conditions) and submitted for protein identification by mass spectrometry. ApoA1 were identified in bands corresponding to both apoA1 and hmapoA1, as illustrated in Fig 1 (lane 10). Our results confirm the presence of N-glycosylation of hm-apoA1 in patients with AMI, as described by Cubedo et al. The results further show that these N-glycosylated hm-apoA1 can be detected in other cardiovascular diseases (UAP and SAP) and in healthy donors. However, we have also found that N-glycosylated hm-apoA1 patterns vary among the different groups,
in particular, between the acute coronary syndrome patients (AMI and UAP) compared with the SAP patients and the control group. This difference is very likely related to the qualitative properties of hm-apoA1, that is, to the site(s) of glycosylation and to the carbohydrate chains’ composition. We are confident that these observations will be very important and should be followed up by precise hm-apoA1 characterization and validation. ACKNOWLEDGMENTS
Conflicts of interest: All authors have read the journals policy on disclosure of potential conflicts of interest and have none to declare. This study was supported by the Czech Science Foundation P205/12/G118 and the Ministry of Health of the Czech Republic project for the conceptual development of the research organization (Institute of Hematology and Blood Transfusion). Pavel Majeka,* Klara Pecankovaa Martin Malyb Milan Oravecc Tomas Riedela Jan E. Dyra a Institute of Hematology and Blood Transfusion Prague, Czech Republic
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Central Military Hospital 1st Medical Faculty Charles University Prague, Czech Republic c Complex Cardiovascular Center for Adult Patients Cardiology Clinic 2nd Faculty of Medicine Charles University and University Hospital Motol Prague, Czech Republic REFERENCES
1. Cubedo J, Padr o T, Badimon L. Glycoproteome of human apolipoprotein A-I: N-and O-glycosylated forms are increased in
2.
3.
4.
5.
patients with acute myocardial infarction. Transl Res 2014;164: 209–22. Majek P, Reicheltova Z, Suttnar J, et al. Plasma proteome changes in cardiovascular disease patients: novel isoforms of apolipoprotein A1. J Transl Med 2011;9:84. Majek P, Reicheltova Z, Stikarova J, Suttnar J, Sobotkova A, Dyr JE. Proteome changes in platelets activated by arachidonic acid, collagen, and thrombin. Proteome Sci 2010; 8:56. Zielinska DF, Gnad F, Wisniewski JR, Mann M. Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints. Cell 2010;141:897–907. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 2012;9: 671–5.
Reprint requests: Pavel Majek, Institute of Hematology and Blood Transfusion, U Nemocnice 1, 128 20 Prague 2, Czech Republic; e-mail: [email protected] Submitted for publication August 7, 2014; accepted for publication September 6, 2014. Translational Research 2015;165:360–362. 1931-5244/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2014.09.003
360
Fig 1. Western blot analysis. Pooled plasma samples from patients with SAP, UAP, and AMI were used together with control samples (C) for a deglycosylation assay (1 or 2 indicate the addition of PNGase F). Lane 10 illustrates sodium dodecyl sulfate–polyacrylamide gel electrophoresis (nonreducing conditions) of the isolated apoA1. Asterisks indicate bands containing apoA1, as identified by mass spectrometry. apoA1, apolipoprotein A1; AMI, acute myocardial infarction; SAP, stable angina pectoris; UAP, unstable angina pectoris; Mw, molecular weight; PNGase F, peptide-N-glycosidase F.
peptide-N-glycosidase F (G1549; Sigma-Aldrich, St. Louis, Missouri). The results are illustrated in Fig 1. It is obvious that hm-apoA1 were detected in the UAP and AMI groups (lanes 6 and 8), weakly detected in SAP (lane 4), and almost not detected in the control group (lane 2), which is in accordance with our previous observation. Herein, we show that after deglycosylation, 2 additional lowered molecular weight bands were detected (50 and 35 kDa); these bands were observed in all the groups with almost equal intensities (lanes 3, 5, 7, and 9). These results show that the hm-apoA1 are N-glycosylated and, in particular, are presented in all the studied groups as estimated by deglycosylation. Moreover, the results show that the N-glycosylation pattern of hm-apoA1 (the degree and position of N-glycosylation) is very likely altered among the groups. The presence of glycosylation restricts the interaction of the antibody with the hm-apoA1, which results in limited detection (lanes 2 and 4), yet the bands can still be detected after deglycosylation (lanes 3 and 5). To highlight the changes observed and to make the results
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Fig 2. Comparison of band intensities. The band intensities were compared before and after deglycosylation (the positions of the bands are indicated in (C) using ImageJ software (A). A relative comparison of the same bands is illustrated in (B). AMI, acute myocardial infarction; SAP, stable angina pectoris; UAP, unstable angina pectoris; PNGase F, peptide-N-glycosidase F.
comparable, our data were further processed with ImageJ software.5 Fig 2, A shows the relative comparison of band intensities before and after deglycosylation (intensities were normalized to the total apoA1 level). For better clarity, the same bands are presented normalized with respect to the control group (Fig 2, B). The differences in intensity of hm-apoA1 among the studied groups are obvious when compared with the deglycosylated hm-apoA1. To eliminate the possibility of a nonspecific antibody interaction (false detection), apoA1 and hm-apoA1 were isolated using a Dynabeads Protein G immunoprecipitation kit (Invitrogen, Carlsbad, California) according to the manufacturer’s instructions from pooled UAP and AMI samples, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (nonreducing conditions) and submitted for protein identification by mass spectrometry. ApoA1 were identified in bands corresponding to both apoA1 and hmapoA1, as illustrated in Fig 1 (lane 10). Our results confirm the presence of N-glycosylation of hm-apoA1 in patients with AMI, as described by Cubedo et al. The results further show that these N-glycosylated hm-apoA1 can be detected in other cardiovascular diseases (UAP and SAP) and in healthy donors. However, we have also found that N-glycosylated hm-apoA1 patterns vary among the different groups,
in particular, between the acute coronary syndrome patients (AMI and UAP) compared with the SAP patients and the control group. This difference is very likely related to the qualitative properties of hm-apoA1, that is, to the site(s) of glycosylation and to the carbohydrate chains’ composition. We are confident that these observations will be very important and should be followed up by precise hm-apoA1 characterization and validation. ACKNOWLEDGMENTS
Conflicts of interest: All authors have read the journals policy on disclosure of potential conflicts of interest and have none to declare. This study was supported by the Czech Science Foundation P205/12/G118 and the Ministry of Health of the Czech Republic project for the conceptual development of the research organization (Institute of Hematology and Blood Transfusion). Pavel Majeka,* Klara Pecankovaa Martin Malyb Milan Oravecc Tomas Riedela Jan E. Dyra a Institute of Hematology and Blood Transfusion Prague, Czech Republic
362
Translational Research February 2015
jek et al Ma b
Central Military Hospital 1st Medical Faculty Charles University Prague, Czech Republic c Complex Cardiovascular Center for Adult Patients Cardiology Clinic 2nd Faculty of Medicine Charles University and University Hospital Motol Prague, Czech Republic REFERENCES
1. Cubedo J, Padr o T, Badimon L. Glycoproteome of human apolipoprotein A-I: N-and O-glycosylated forms are increased in
2.
3.
4.
5.
patients with acute myocardial infarction. Transl Res 2014;164: 209–22. Majek P, Reicheltova Z, Suttnar J, et al. Plasma proteome changes in cardiovascular disease patients: novel isoforms of apolipoprotein A1. J Transl Med 2011;9:84. Majek P, Reicheltova Z, Stikarova J, Suttnar J, Sobotkova A, Dyr JE. Proteome changes in platelets activated by arachidonic acid, collagen, and thrombin. Proteome Sci 2010; 8:56. Zielinska DF, Gnad F, Wisniewski JR, Mann M. Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints. Cell 2010;141:897–907. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 2012;9: 671–5.
