The appendix-specific proteome
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The appendix is a blind-ended short intestinal protrusion extending from the large intestine's cecum, and as the shape resembles a worm, thus often referred to as "appendix vermiformis". The histology of the appendix resembles the histology of the colon, with the four layers of mucosa, submucosa, muscularis externa and serosa. Unlike the large intestine, however, the submucosa of the appendix contains nodules of lymphoid tissue. The transcriptome analysis shows that 71% of all human proteins (n=19628) are expressed in the appendix and 205 of these genes show an elevated expression in appendix compared to other tissue types.
An analysis of the genes with elevated expression in appendix, with regards to function, reveals that they are dominantly involved in immune system processes.
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- 2 appendix enriched genes
- 205 genes defined as elevated in the appendix
- Most of the elevated genes in appendix encode proteins involved in immune system processes
- Most group-enriched genes are shared with lymph node
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Figure 1. The distribution of all genes across the five categories based on transcript abundance in appendix as well as in all other tissues.
205 genes show some level of elevated expression in the appendix compared to other tissues. The three categories of genes with elevated expression in appendix compared to other organs are shown in Table 1.
Table 1. Number of genes in the subdivided categories of elevated expression in appendix
Category |
Number of genes |
Description |
| Tissue enriched |
2 |
At least five-fold higher mRNA levels in a particular tissue as compared to all other tissues |
| Group enriched |
51 |
At least five-fold higher mRNA levels in a group of 2-7 tissues |
| Tissue enhanced |
152 |
At least five-fold higher mRNA levels in a particular tissue as compared to average levels in all tissues |
| Total |
205 |
Total number of elevated genes in appendix |
Protein expression of genes elevated in appendix
The list of elevated genes (n=205) are well in-line with the function of the appendix, as it includes an overrepresentation of proteins associated with immune system processes. Among the genes elevated only in appendix (n=154) there is an overrepresentation of proteins involved in response to stimulus and chemotaxis, indicative of an active inflammation. CXCR1 and CXCR2, two receptors for IL-8 (also known as neutrophil chemotactic factor), both show an enhanced expression in appendix. IL-8 induces chemotaxis in neutrophils, and thereby neutrophils are attracted to the site of infection. An inflammatory reaction pattern could reflect that appendix samples were obtained from cases with some degree of appendicitis.
There were only two genes in the category of genes with tissue enriched expression in the appendix (IRG1 and TNFRSF6B). This is not unexpected as the included cell types, function and morphological features of the appendix are highly similar to other related tissue types, such as the lymph node, tonsil and spleen. Genes that specifically signify these types of tissues will thus be categorized as group enriched genes (see below).
The appendix transcriptome
An analysis of the expression levels of each gene makes it possible to calculate the relative mRNA pool for each of the categories. The analysis shows that 86% of the mRNA molecules in the appendix corresponds to housekeeping genes and only 2% of the mRNA pool corresponds to genes categorized to be either appendix enriched, group enriched, or enhanced. Thus, most of the transcriptional activity in the appendix relates to proteins with presumed housekeeping functions as they are found in all tissues and cells analyzed.
Proteins specifically expressed in neutrophils
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One of the most highly expressed genes in the appendix is FPR1, which is also highly expressed in bone marrow. FPR1 encodes a G protein-coupled receptor protein expressed by e.g. neutrophils, and plays a role in chemotaxis, phagocytosis and generation of reactive oxygen species. The immunohistochemical staining shows strong positivity in a cell population indicative of phagocytes in bone marrow, appendix, as well as in many other tissues.
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Proteins specifically expressed in peripheral blood leukocytes
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The FCN1 gene shows an elevated expression in appendix and bone marrow. M-ficolin encoded by the gene FCN1 is predominantly expressed in peripheral blood leukocytes, and has been postulated to function as a plasma protein with elastin-binding activity.
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Proteins specifically expressed in endothelial cells
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The VIP gene, vasoactive intestinal peptide, encodes for a 28-amino-acid polypeptide,
which stimulates the secretion of electrolytes and water by the intestinal mucosa.
The VIP gene shows an elevated expression in appendix, colon, rectum, smooth muscle and small intestine,
and immunohistochemical staining reveals distinct positivity in blood vessel endothelial cells.
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Proteins specifically expressed in B-cells
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The lymphoid nodules of the appendix constitute dense collections of B-lymphocytes, as shown by the elevated expression of the MS4A1 gene in appendix, spleen and lymph node. The protein CD20 encoded by the MS4A1 gene is an activated-glycosylated phosphoprotein expressed on the surface of B-cells beginning at the pro-B phase with progressively increasing concentrations until maturity.
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Genes shared between appendix and other tissues
There are 51 group-enriched genes expressed in the appendix. Group enriched genes are defined as genes showing a 5-fold higher average level of mRNA expression in a group of 2-7 tissues, including appendix, compared to all other tissues.
In order to illustrate the relation of appendix tissue to other tissue types, a network plot was generated, displaying the number of commonly expressed genes between different tissue types.
Figure 2. An interactive network plot of the appendix enriched and group enriched genes connected to their respective enriched tissues (grey circles). Red nodes represent the number of appendix enriched genes and orange nodes represent the number of genes that are group enriched. The sizes of the red and orange nodes are related to the number of genes displayed within the node. Each node is clickable and results in a list of all enriched genes connected to the highlighted edges. The network is limited to group enriched genes in combinations of up to 4 tissues, but the resulting lists show the complete set of group enriched genes in the particular tissue.
The network plot shows that most genes are shared with lymph node, although most genes shared with lymph node are also shared with other tissues harboring a major component of lymphoid cells, i.e. tonsil and spleen.
Appendix function
It is well accepted that the immune tissue called gut associated lymphoid tissue (GALT) is important for fighting pathogens passing through the glandular epithelium of the gut. However, the function of the appendix is much debated due to the apparent lack of importance, as judged by an absence of side effects following appendectomy. One hypothesis is that the appendix constitutes a vestigial remnant of a once larger cecum, while another hypothesis suggests that it acts as storage for beneficial bacteria during times of illness.
Figure 3. Schematic view of the appendix. Attribution: By Mariana Ruiz Villarreal (LadyofHats) (Own work) [Public domain], via Wikimedia Commons. Source. Image has been cropped.
The histology of human appendix including detailed images and information can be viewed in the Protein Atlas Histology Dictionary.
Background
Here, the protein-coding genes expressed in the appendix are described and characterized, together with examples of immunohistochemically stained tissue sections that visualize protein expression patterns of proteins that correspond to genes with elevated expression in the appendix.
Transcript profiling and RNA-data analyses based on normal human tissues have been described previously (Fagerberg et al., 2013). Analyses of mRNA expression including over 99% of all human protein-coding genes was performed using deep RNA sequencing of 172 individual samples corresponding to 37 different human normal tissue types. RNA sequencing results of 3 fresh frozen tissues representing normal appendix was compared to 169 other tissue samples corresponding to 36 tissue types, in order to determine genes with elevated expression in appendix. A tissue-specific score, defined as the ratio between mRNA levels in appendix compared to the mRNA levels in all other tissues, was used to divide the genes into different categories of expression.
These categories include: genes with elevated expression in appendix, genes expressed in all tissues, genes with a mixed expression pattern, genes not expressed in appendix, and genes not expressed in any tissue. Genes with elevated expression in appendix were further sub-categorized as i) genes with enriched expression in appendix, ii) genes with group enriched expression including appendix and iii) genes with enhanced expression in appendix.
Human tissue samples used for protein and mRNA expression analyses were collected and handled in accordance with Swedish laws and regulation and obtained from the Department of Pathology, Uppsala University Hospital, Uppsala, Sweden as part of the sample collection governed by the Uppsala Biobank. All human tissue samples used in the present study were anonymized in accordance with approval and advisory report from the Uppsala Ethical Review Board.
Relevant links and publications
Uhlén M et al, 2015. Tissue-based map of the human proteome. Science
PubMed: 25613900 DOI: 10.1126/science.1260419 Yu NY et al, 2015. Complementing tissue characterization by integrating transcriptome profiling from the Human Protein Atlas and from the FANTOM5 consortium. Nucleic Acids Res.
PubMed: 26117540 DOI: 10.1093/nar/gkv608 Fagerberg L et al, 2014. Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol Cell Proteomics.
PubMed: 24309898 DOI: 10.1074/mcp.M113.035600 Andersson S et al, 2014. The transcriptomic and proteomic landscapes of bone marrow and secondary lymphoid tissues. PLoS One.
PubMed: 25541736 DOI: 10.1371/journal.pone.0115911 Histology dictionary - appendix
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