The salivary gland-specific proteome

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Figure 1. The distribution of all genes across the five categories based on transcript abundance in salivary gland as well as in all other tissues.

86 genes show elevated expression in the salivary gland compared to other tissues. The three categories of genes with elevated expression in salivary gland compared to other organs are shown in Table 1.

Table 2. The 12 genes with the highest level of enriched expression in salivary gland. "Predicted localization" shows the classification of each gene into three main classes: Secreted, Membrane, and Intracellular, where the latter consists of genes without any predicted membrane and secreted features. "mRNA (tissue)" shows the transcript level as�TPM values,�TS-score (Tissue Specificity score) corresponds to the score calculated as the fold change to the second highest tissue.

Some of the proteins predicted to be membrane-spanning are intracellular, e.g. in the Golgi or mitochondrial membranes, and some of the proteins predicted to be secreted can potentially be retained in a compartment belonging to the secretory pathway, such as the ER, or remain attached to the outer face of the cell membrane by a GPI anchor.

The salivary gland 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 almost half (54%) of the mRNA molecules in the salivary glands correspond to genes categorized to be either salivary gland enriched, group enriched or, salivary gland enhanced, whereas 41% of the mRNA is derived from housekeeping genes expressed in all tissues.

Gene Ontology-based analysis of all the 86 genes elevated in salivary gland indicates a clear overrepresentation of proteins associated with body fluid secretion and biomineral tissue development. A majority of the 86 genes encodes for secreted proteins.

Protein expression of genes elevated in salivary gland

In-depth analysis of the elevated genes in salivary gland using antibody-based protein profiling allowed us to create a map of where these proteins are expressed within the salivary gland with respect to serous or mucinous cells secretion and expression in ductal epithelial cells.

Proteins specifically expressed in serous salivary glands

Serous salivary glands are found mainly in the parotid gland and secrete granules that are rich in proteins that have high amylase activity. Four examples of proteins expressed in serous salivary glands are CA6, CST2 and PIP and AMY1B.

Proteins specifically expressed in mucinous salivary glands

Mucinous salivary glands are found mainly in the sublingual gland and are rich in glycoproteins. One example of a protein specific for mucinous salivary glands is MUC7, which has a protective capacity by promoting clearance of bacteria in the oral cavity, as well as aiding in mastication, speech and swallowing.

Proteins specifically expressed in salivary ducts

The ducts of the salivary gland are essential for passage of the saliva from the glands to the oral cavity. They also have ion-pumping activity, modifying the composition of the secretion from the acinar cells. One examples of a protein expressed in salivary ducts is SLC5A5, which is important for iodine uptake in the thyroid but with unknown function in salivary gland.

Genes shared between salivary gland and other tissues

There are 24 group enriched genes expressed in the salivary gland. 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 salivary gland, compared to all other tissues.

In order to illustrate the relation of salivary gland 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�salivary gland�enriched and group enriched genes connected to their respective enriched tissues (grey circles).�Red�nodes represent the number of�salivary gland 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�3�tissues, but the resulting lists show the complete set of group enriched genes in the particular tissue.

Pancreas is an organ with exocrine function highly correlated with the salivary gland. One example of a group enriched gene shared between pancreas and salivary gland is BHLHA15, a transcription factor regulating acinar cell function.

Salivary gland function

The main function of the salivary glands is to wet and lubricate the oral cavity and its contents, in order to initiate digestion of carbohydrates. Saliva also has an important buffer function and plays a role in taste. Moreover, salivary glands are involved in the defense against microorganisms, secreting various protective substances. There are three pairs of major salivary glands: parotid glands, submandibular (submaxillary) glands and sublingual glands. In addition to the main salivary glands, 600-1,000 minor mucous secreting glands are present in the palate, nasal and oral cavity.

Salivary gland histology

The major salivary glands are the paired parotid, sublingual and submandibular glands. They produce saliva, a mixture of serous and mucinous secretions containing water, proteins, glycoproteins and electrolytes, secreted into the oral cavity. Saliva is rich in enzymes that initiate the breakdown of the food we eat and lubricates ingested food to facilitate swallowing.

The salivary glands are surrounded by connective tissue capsules, which also divide the gland into lobes and lobules and contain larger arteries and veins. The salivary glands are tubuloacinar glands, with branched ducts ending in sac like dilations (acini) where the excretory cells are located. The secretory unit of the salivary gland is the salvion, consisting of an acinus, intercalated duct, striated duct and excretory duct.

The acini can be of serous, mucous or mixed type. The serous acini secrete a fluid that is rich in proteins, with triangular shaped cells that appear darkly stained with hematoxylin-eosin (HE) and have basally located nucleus. The basal portion of the glands appear darker stained due of the presence of rough endoplasmic reticulum, while the apical portion stains lighter due the presence of secretory granules. Mucous acini can easily be distinguished from serous acini, as the cells of the mucous acini are paler and contain flattened nuclei located towards the base of these cells.

Intercalated ducts, with low cuboidal epithelium, lead away from the acini. In serous secreting glands these small ducts are more evident and secrete bicarbonate and absorb chloride ion from the acinar secretions.

Striated ducts connect the intercalated ducts with the larger excretory ducts. They are lined with simple cuboidal or simple columnar epithelium, depending on the size of the duct. They are termed striated ducts because the basal plasma membrane folds into the lower portion of the cell, resulting in a striated appearance. The nuclei are spherical and located in the centre of the cell.

Excretory ducts are located in the interlobular and interlobar connective tissue. As the diameter of the ducts increase, the epithelium changes from simple cuboidal to pseudo-stratified columnar or stratified cuboidal. Excretory ducts eventually open into the oral cavity.

Within the salivary glands there are also aggregations of adipocytes. Fat is stored in adipocytes as a single large lipid droplet. In routine embedding of histological specimens the lipid is dissolved, leaving a large unstained and empty appearing space within each adipocyte. The nucleus is flattened and displaced by the lipid droplet to the periphery of the cell.

The histology of human salivary gland including detailed images and information about the different cell types can be viewed in the Protein Atlas Histology Dictionary.

Background

Here, the protein-coding genes expressed in the salivary gland 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 salivary gland.

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 salivary gland was compared to 169 other tissue samples corresponding to 36 tissue types, in order to determine genes with elevated expression in salivary gland. A tissue-specific score, defined as the ratio between mRNA levels in salivary gland 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 salivary gland, genes expressed in all tissues, genes with a mixed expression pattern, genes not expressed in salivary gland, and genes not expressed in any tissue. Genes with elevated expression in salivary gland were further sub-categorized as i) genes with enriched expression in salivary gland, ii) genes with group enriched expression including salivary gland and iii) genes with enhanced expression in salivary gland.

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

Histology dictionary - the salivary gland