Glycoproteomic analysis of plasma from patients with atopic dermatitis: CD5L and ApoE as potential biomarkers

Abstract

Atopic dermatitis (AD) is an inflammatory skin disorder that is both uncomfortable and distressing to patients, and its prevalence has been steadily increasing. It is obvious that the identification of efficient markers of AD in plasma would offer the possibility of effective diagnosis, prevention, and treatment strategies. In this study, a proteomic approach was used to analyze plasma glycoproteins from both children with AD and healthy child donors. Several protein spots showing significant quantitative changes in the AD patients were identified. Through sequential studies, it was confirmed that CD5L and ApoE were significantly up-regulated or down-regulated, respectively, in the plasma from AD patients compared with that from healthy donors. In addition, we suggest that the up-regulated CD5L in AD patients causes eosinophilia by inhibiting apoptosis or promoting the proliferation of eosinophils either in combination with or without IL-5. The glycoproteomic data in this study provides clues to understanding the mechanism of atopic alterations in plasma and suggests AD-related proteins can be used as candidate markers for AD.

Figure 1

Representative LCA lectin blot analysis of plasma from healthy control and AD patient. Total plasma proteins extracted from the healthy donors and the AD patients were separated on 2-DE gels and transferred onto a PVDF membrane. The membrane was subjected to a lectin blot and treated with biotin-labeled LCA. Spots that displayed differently (marked by circles) were detected.

Figure 2

The analysis of AD plasma using 2-DE. (A) Representative 2-DE map of plasma from an AD patient. The differentially expressed protein spots are indicated by a circle or rectangle. The results are summary of 2-DE results from all AD patients and normal control. The spots indicated were identified using either MALDI-TOF or LC-MS/MS, as outlined in Table 2. (B) Enlargement of the 2-DE gel on up-regulated and down-regulated spots. (C) Quantitation of the proteins shown in (B). The control volume is expressed as 100% and the volumes of the AD patients as values relative to the control. The data represent the mean values ± SD. ^*P < 0.05; ^**P < 0.01.

Figure 3

Validation of the ApoE expression level via Western blotting. (A) Western blot analyses of serum protein (5 µl) from four healthy donors and six AD patients were conducted. (B) Quantitation analysis of the gel bands was conducted using Image J software. Significant differences (P < 0.05) were measured via a median plus ANOVA.

Figure 4

Validation of the CD5L expression level and effect of CD5L on AML14.3D10 proliferation. (A) Western blot analysis of the serum protein (5 µl) from six healthy donors and six AD patients were conducted. Quantitation analyses of the gel bands were conducted using Image J software. Significant differences (P < 0.01) were measured via a median plus ANOVA. (B) CD5L increases the proliferation of the AML14.3D10 cells. AML14.3D10 cells were seeded into 96 well plates and grown for 24 h. The cells were then treated with CD5L (± IL-5 or Dexamethasone). At each time point, the cell viability was measured. (C) CD5L induces the proliferation of AML14.3D10 cells in a dose-dependent manner in the presence of IL-5. (D) CD5L induces the proliferation of AML14.3D10 cells in a dose-dependent manner in the presence of Dexamethasone.