The Role of Sorting Nexins in Antigen Presentation Chng X.R.J 1 and Wong S.H. 2 Department of Microbiology Yong Loo Lin School of Medicine, National University of Singapore Block MD4, 5 Science Drive 2, Singapore 11759 ABSTRACT The Sorting Nexins (SNX) are a family of proteins that possess a phoxhomology (PX) domain and which may possess sorting functions. It has been shown in recent studies that some SNX have a role to play in the retromer, a complex that rescues receptors from destruction in the lysosomes and transports these receptors to the golgi. This paper postulates that there may be a link between SNX 5 and 6 and antigen presentation, particularly in the degradation of antigens. However, due to time constraints, this paper only presents the basic groundwork that was laid that can act as a platform to test this postulate in the near future. INTRODUCTION The Sorting Nexin (SNX) Family is a group of cytoplasmic, membraneassociated proteins that are involved in endocytosis and protein trafficking in the cell 1,2,. These SNXs possess a phox-homology (PX) domain that binds to phosphotidylinositol phosphates 1,2,3. More recently, studies have shown that some SNX may be involved with the retromer, a complex that recycles receptors from the endosomes to the golgi, effectively rescuing these receptors from destruction in the lysosomes. The yeast retromer is well studied and consists of Vps35, Vps26, Vps29p and a Vps5p-Vps17 dimer. The former three components have a role in structural identification, where as 1 Student 2 Supervisor
the dimer has a structural role in membrane deformation. It was subsequently found that SNX 1 is the mammalian ortholog of Vps5p 2,3,4,5. It was also recently postulated that SNX 5 and SNX 6 may be involved with the retromer. When tested, it was found that repressing SNX 5 and 6 resulted in a phenotype similar to that of repressing known retromer constituents. Also, it was noted that SNX 5 and 6 could co-localise with SNX 1; and more remarkably, the repression of SNX 5 and 6 induced a loss of SNX 1. The paper concluded that SNX 5 and 6 might be an ortholog for Vps17 5. SNX 1 is a relatively well-studied member of the SNX family and it was discovered that it has a role in antigen presentation. It was initially found that Shiga toxin was partially transported to the golgi via SNX 1-labelled endosomes, and that when SNX 1 was suppressed in the cell, Shiga toxin was not as effectively transported. However, the transport of Shiga toxin was not exclusive to SNX 1- labelled endosomes 7. As such, since SNX 1 has been demonstrated to be part of the retromer, and since SNX 5 and 6 have probable roles in the retromer, there might be a likely link between SNX 5 and 6 and antigen presentation. As the retromer is involved in endosome-to-golgi transport, there is a possibility that SNX 5 and 6 may be involved in the trafficking of antigens to the lysosome for degradation during antigen processing, an essential part of antigen presentation. One way of assaying the impact of SNX 5 and 6 on antigen degradation is to monitor the effect SNX 5 and SNX 6 over-expression has on the distribution and degradation of scavenger receptors such as CD204 in macrophages. CD204 is of interest as it is a receptor specific for the uptake of Escherichia coli by macrophages. The qualitative and quantitative assays involving CD204 internalisation were set up, and the endogenous expression of SNX 5 and 6 in RAW cells was studied and attempts were made to overexpress them. MATERIALS AND METHODS The methods used in this study were western blot and immunofluorescence. Western blot was employed to quantitate the internalisation of CD204, while
immunofluorescence was used to visualise the internalisation of CD204 and depict endogenous SNX 5 and 6 expression. Transfection methods were used to attempt to increase overexpress the SNX 5 and 6 levels within RAW cells. Immunofluorescence Rat anti-mouse CD204 antibodies from AbD Serotec and goat anti-mouse antibodies for SNX 5 and 6 were used in these assays. The RAW cells were fixed with 4% paraformaldehyde and permeabilised with 0.1% Saponin or 0.1% Triton-X. Following this, RAW cells were incubated in primary antibody and subsequently in fluorescent-labelled secondary antibody. The slides were mounted in medium containing DAPI and subsequently viewed. Transfection to overexpress SNX 5 and 6 Transfection was carried out using Lipofectamine 2000 TM and Effectene TM with differing concentrations of DNA (Lipofectamine 2000 TM : 4ug and 8ug of DNA; Effectene TM : 0.4ug, 0.8ug, 1.6ug, 3.2ug, 4.8ug, 6.4ug) in RAW cells. Effectene TM was purchased from QIAGEN, and Lipofectamine 2000 TM from Invitrogen. Western blotting The antibody that was used in this assay was a rat anti-mouse CD204 antibody from AbD Serotec. RESULTS AND DISCUSSION An assay system to qualify and quantify the internalisation of CD204 was constructed. As we expect the overexpression of SNX 5 and 6 to have an impact on the internalisation of CD204, this platform is essential to assessing the effects the overexpression of SNX 5 and 6 on CD204 internalisation. SNX 5 and 6 were observed and efforts were made to over express them; however, the latter step proved problematic and unsuccessful. Quantitative analysis of CD204 internalisation This kinetics experiment was conducted using western blot at time points of 0, 5, 10, 15, 30 and 60 minutes on RAW cells with an anti-cd204 antibody. It was
observed that there was rapid degradation of CD204, with discernable difference being noted five minutes after the commencement of the experiment. This assay can be used on RAW cells overexpressing SNX 5 and 6 to determine the effect, if any, that SNX 5 and 6 have on the degradation of CD204. Qualitative analysis of CD204 internalisation A kinetics experiment involving two time points (0 min and 60 min) was carried out to observe the internalisation of CD204 and an attempt to co-localise CD204 with VAMP 3, and early endosome marker was also done. It was observed that CD204 on the surface was transported into endosomes after an hour, with no colocalisation with VAMP 3 i.e. CD204 does not reside in early endosomes an hour after internalisation. More investigations can be done on this experiment, for example, co-localisation with other markers e.g. VAMP 8, and it could be repeated at more time points to more clearly see the internalisation. Endogenous expression of SNX 5 and 6 SNX 5 and 6 were observed, and co-localisation of SNX 6 with vti1a was carried out. SNX 5 was not co-localised with vti1a as the antibodies available were not compatible. Vti1a is a marker for endosomes and the golgi 6,7. It was noted that there is co-localisation between SNX 6 and vti1a, and it seems that both SNX 5 and 6 are predominantly expressed in the endosomes, reflecting what was previously reported in the literature. Transfection of SNX 5 and 6 SNX 5 and 6 were overexpressed transiently in RAW cells by transfection with plasmid DNA. However, the transfection efficiencies remained low, despite changing various parameters like the reagents used, the amount of DNA added and the purity of the DNA. The experiment was unable to proceed from there, but to circumvent this problem, a stable clone that overexpresses SNX 5 or 6 can be selected and obtained. REFERENCES
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