Comparison of camel tear proteins between summer and winter

Abstract

Purpose: Proteins in the tear fluid have positive effects on maintaining the integrity and stabilization of the tear film, which is affected by several environmental factors. The aim of this study is to investigate seasonal variation of protein patterns in camel tears collected during the summer and winter season.

Methods: Tears from both eyes of 50 clinically normal camels (Camelus dromedarius) were collected in the summer (June-July) and in the winter (December-January) respectively. Pooled tear protein samples from two seasons were separated by SDS-PAGE and two-dimensional electrophoresis (2-DE). Protein spots of differential expression in two season gels were excised and subjected to in-gel digestion and identification by matrix assisted laser desorption/ionization-time of flight/time of flight-mass spectrum (MALDI-TOF/TOF-MS) analysis. Two differentially expressed proteins, lactoferrin (LF) and vitelline membrane outer layer protein 1 homolog (VMO1 homolog), were validated by western blotting.

Results: Thirteen well resolved bands were detected in SDS-PAGE gels of both summer and winter camel tears. By band densitometry, significantly higher intensities of band 6, 7, 11, and lower intensity of band 13 were observed in the summer group compared to the winter group. In 2-DE profiles of camel tears, four protein spots were found expressed differentially in two seasons. Further protein identification by MALDI-TOF/TOF-MS and confirmation by western blotting indicated that there was a significant decrease in LF (p=0.002) and an increase in VMO1 homolog (p=0.042) in tears in the summer compared to the winter.

Conclusions: The seasonal variation of camel tear fluids has been found in the composition of proteins, including LF and VMO1 homolog. This result will expand our knowledge of physiologic characteristics of tear fluids and establish a foundation for the mechanistic studies and clinical practices on ocular surface disorders.

Figure 1

Comparison of SDS–PAGE gel patterns of proteins in camel tear fluids between summer and winter. A: Proteins of camel tears in the summer (lane Cs) and in the winter (lane Cw) were separated on a 13% gel with equal amount of total tear proteins in each sample. Thirteen well resolved bands are detected in both lanes. B: Graphic of lane comparison of camel tear proteins between the summer (dotted line) and the winter (solid line). B1, Band1; B2, Band2; B3, Band3; and so forth in B are correspondent with those in A.

Figure 2

Comparison of 2-DE Coomassie-stained protein profiles and differential expression spots of camel tears between summer and winter. A: Tear proteins (100 μg) in the summer (Cs) and in the winter (Cw) were separated on first-dimensional pH 3–10 linear IPG gels (13 cm) and second-dimensional 13% vertical slab gels. The relative MW is given on the left, while the pI is given at the top of the figure. The spots marked by arrows and numbers were cut and digested, and then identified using MALDI-TOF/TOF-MS. B-I: Protein spots w1, w2, w3 and s7, and w7 with different volume intensities are displayed in the enlarged spot views of 2-DE images (B-E) and as three-dimensional images obtained by Melanie 4.0 software (F-I). Spots w1, w2, w3, w4, w5, w6 and s4, s5, and s6 were identified as LF and spots s7 and w7 were characterized as VMO1 homolog. B, D, F, H: The summer group (Cs); C, E, G, I: The winter group (Cw).

Figure 3

The MALDI-TOF/TOF mass spectrum analysis of spot w6 in Figure 2 indentified as LF (Camelus dromedarius, gi|5777368). A: The PMF signals. B: The MS/MS spectrum of parent ion 1570.794 for the sequence KPVDAFQECHLAR calculated by b ions (b*) and y ions (y*).

Figure 4

Western blot analysis of decreasing expression of LF and increasing expression of VMO1 homolog in camel tears in the summer compared to the winter. A, B: Comparison of expression of LF (A) and VMO1 homolog (B) between the summer group (Cs) and the winter group (Cw) by western blotting. C, D: Relative quantitative analysis of each corresponding band of LF (C) and VMO1 homolog (D) in two groups, based on the volume intensity of the band in Cw as 1.0. The paired student’s t test was performed and showed a significant difference (*p=0.042, **p=0.002) between two groups.