Detec%ng Unique Proteins in Rheumatoid Arthri%s Pa%ents with Inters%%al Lung Disease
Introduction Rheumatoid Arthritis is a systemic autoimmune disease which primarily causes in5lammation in joint tissues. Other tissues of the body are affected by this disease as well though, such as lung tissue, which was the focus of this study. In5lammation in the lungs caused by RA can lead to Interstitial Lung Disease (ILD), a broad array of complications which affect 10-‐20% of RA patients and can be fatal. Currently, the cause of the autoimmune response in the lung tissues is unknown, but it has been hypothesized that citrullinated proteins (proteins that have had the amino acid arginine converted to citrulline by the enzyme peptidylarginine deiminase) could represent the basis of this autoimmune response. While the underlying origin of protein citrullination in the lungs is not fully understood either, these proteins nevertheless represent a potentially inexpensive and noninvasive biomarker that could be used in early detection of RA-‐ILD.
Question Do RA patient with ILD possess unique citrullinated protein pro5iles compared to patients without ILD?
Alexander Davis Hurley1, Phu Van1, Dana Ascherman, M.D.2, 3, John Minden1 1Department of Biological Sciences, Carnegie Mellon University
2Division of Rheumatology, University of Miami 3Miami VA Medical Center
Figure 1. An image of a chest x-ray of a patient with idiopathic interstitial pneumonia – a disease that is considered a part of the Interstitial Lung Diesease category.
http://casereports.bmj.com/content/2010/bcr.02.2010.2765/F1.large.jpg
Materials Blood serum samples were acquired from the two subgroups of patients (RA-‐No ILD and RA-‐ILD). The RA-‐No ILD category included samples 15 and 16, and the RA-‐ILD category included samples HAW, 27, and 28. While these serum samples did not contain high levels of the proteins in question, they did contain the antibodies produced by the patient’s immune system to target the citrullinated proteins. Because they have high binding speci5icity, and because they are more abundant than the citrullinated proteins, these antibodies were considered to be viable proxies for patient-‐speci5ic protein samples. The citrullinated and non-‐citrullinated proteins used in the experiment were instead produced though in vitro, PAD-‐catalyzed deimination of cell lysates prepared from K562 and various lung-‐derived cell lines (Ascherman).
Methods 1) Binding: • Mix the sample of one patient with Protein A sepharose beads to allow the antibodies in the sample to bind with the beads • Split antibody-‐bead complex into two components • Bind one component with citrullinated protein extract and the other with uncitrullinated protein extract
2) Two Dimensional Difference Gel Electrophoresis: • Minimally label citrullinated samples with one 5luorescent cyanide dye and uncitrullinated samples with another (Cy3 and Cy5) • Combine the two samples together • Isoelectrically focus the mixture on a pH 3-‐10 gel strip • Co-‐electrophorese the pH separated proteins on a polyacrylamide gel
3) Imaging: • Fluorescently image each dye channel of the gel (Cy3 and Cy5) • Combine these pictures into an overlay (see overlay example below) that allows difference proteins to be identi5ied among background protein • Compare this overlay with the overlay from other patients and determine unique differences between categories
Results
Figure 2. This image depicts the citrullination of a protein. The change in protein structure occurs when the oxygen atom replaces the nitrogen and the hydrogen located at the top right of the structure depicted here.
http://upload.wikimedia.org/wikipedia/commons/thumb/b/b6/Citrullination.svg/400px-‐Citrullination.svg.png
Blood Serum Sample
Mix with Citrullinated
Protein Extracts
Mix with Uncitrullinated
Protein Extracts
Figure 3. This 5low chart outlines the binding and 2D-‐DIGE steps of the experimental protocol. This diagram was inspired by a similar 5igure in Gong, et. al.’s Drosophila Ventral Furrow Morphogenesis: A Proteomic Analysis. 2004.
Conclusions and Future Work This project represents one of the 5irst attempts to analyze the blood serum of these Rheumatoid Arthritis patients with the 2D-‐DIGE technique. Because of this, it is dif5icult to conclude with certainty that the differences detected between the RA-‐ILD and RA-‐No ILD categories represent reliable indicators of the disease complication. Looking at the data gathered though, one must conclude that the ILD category did not have unique citrullinated spots that distinguished these patient from those with RA-‐No ILD. However, it did appear that the RA-‐No ILD category possessed more unique uncitrullinated or mixed spots than the other category. Perhaps the absence of these spots in the RA-‐ILD category could represent an important indicator of the complication. To know for sure, several aspects of the experimental design could be altered or otherwise improved upon in order to more effectively answer the initial question of the project. First, removing the antibodies from the 5inal protein mixture prior to isoelectric focusing and electrophoresis would signi5icantly clear up background 5luorescence in the gel images. With less background clutter in the image it is possible that more difference proteins could be identi5ied between the RA-‐ILD and RA-‐No ILD samples used in this experiment. Another alteration to the experimental design of particular interest is the co-‐electrophoresing of samples from different patients. For example, sample 16 and sample HAW (No-‐ILD and ILD categories, respectively) could both be combined with citrullinated proteins, labeled, and then co-‐electrophoresed. This method would allow for an even more direct comparison of the citrullinated protein pro5iles of the patients involved in the study.
Acknowledgements I am indebted to the Minden lab, the NSF-‐REU coordinators at Carnegie Mellon, the National Science Foundation, Centre College, and the James G. Brown Foundation for all the support this summer.
References Ascherman, D., et. al., Peripheral Blood Biomarkers of Rheumatoid Arthritis-‐Associated Interstitial Lung Disease. 2010.
No-‐ILD ILD
Sample 16 Sample HAW Sample 27 Sample 28
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2
3
Citrullinated Protein: Green Uncitrullinated Protein: Red Mix of Cit./Uncit. Protein: Yellow
No-‐ILD ILD
Sample 16 Sample HAW Sample 27 Sample 28
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2
3
Discussion • Note: Sample 15, which was in the No-‐ILD category, was removed from 5inal analysis because the gel was poorly isoelectrically focused and could not be accurately compared with other gels.
Table 1 displays three protein groups that are common to both categories of RA patient. • Group number 1 shows two citrullinated protein spots that were common to the upper left hand corner of the gels. • Group number 2 shows a string of citrullinated and uncitrullinated proteins in the upper middle of the gels. • Group number 3 shows a string of citrullinated proteins common to all gels in the middle region of all the gels.
Table 2 displays three protein groups that are unique to one gel or another. • Group 1 of this table shows an uncitrullinated protein spot in sample 16 that is not present in the ILD category at all. • Group 2 shows that sample 16 and HAW, although from patients in different categories, have several prominent citrullinated spots in the lower portion of the gel that are unique from samples 27 and 28. • Group 3 shows a very bright set of mixed (Citrullinated and Uncitrullinated) proteins in samples 16. The ILD gels may display this same set of proteins, but they are very faint when comparing images that are matched for intensity.
Figure 4. An example of a full gel overlay. Pictured here is sample 16.
Table 1. Three Similar Protein Spots Among Both Categories of Patient Table 2. Three Unique Protein Spots
Citrullinated Protein: Green Uncitrullinated Protein: Red
Mix of Cit./Uncit. Protein: Yellow
