The human secretomeDefining the secretomeClassification of the secretomePrediction of transmembrane protein topology and signal peptidesClassification of the human proteome The secretory pathwayRelevant links and publications

Tissue methods

The human secretome

A secreted protein can be defined as a protein that is actively transported out of the cell. In humans, certain cell types, such as endocrine cells and B lymphocytes, are specialized for protein secretion, but all cells in the body secrete proteins to varying degrees. Secreted proteins play fundamental roles in intercellular communication, tissue homeostasis, immune defense, and numerous physiological and pathological processes. Medically important secreted proteins include cytokines, coagulation factors, growth factors, and other signaling molecules. A large fraction of the currently approved clinical treatments use drugs targeting or consisting of secreted proteins. Among the FDA-approved drugs currently on the market (see the Druggable Proteome for more details) there are 854 protein targets with known pharmacological action of which 120 are secreted proteins. Besides being a rich source of new therapeutics and drug targets, a large fraction of blood diagnostic tests used in the clinic target secreted proteins, highlighting the importance of this class of proteins in medicine and biology.

Defining the secretome

The human secretome comprises all proteins that are potentially secreted from the cell. These proteins were identified based on UniProt annotations of subcellular locations, along with computational predictions of signal peptides and transmembrane regions. The signal peptide is found in most secreted proteins, but also appears in some classes of membrane proteins. Therefore, the presence of transmembrane regions can be used to distinguish membrane proteins from secreted proteins. A whole-proteome scan of all Ensembl transcripts was performed using majority decision methods for signal peptide prediction (MDSEC) and membrane region prediction (MDM). Proteins with a predicted signal peptide by MDSEC and no predicted transmembrane region by MDM were considered secreted. Using this combined approach, the analysis predicts that 2528 genes (13% of all human protein-coding genes) encode at least one predicted secreted transcript or have at least one isoform with the subcellular location "Secreted" in UniProt.

Secreted to extracellular matrix Secreted to blood Secreted in other tissues Secreted in brain Secreted - unknown location Secreted to digestive system Secreted in male reproductive system Intracellular and membrane Immunoglobulin genes Secreted in female reproductive system

Figure 1. Classification of the human secretome. More data for the individual categories can be explored by clicking on the icons, and corresponding gene lists are obtained by clicking on the numbers in the pie chart.

Classification of the secretome

The predicted secreted genes were then systematically annotated using a combination of literature curation, bioinformatic analysis, and experimental data from multiple resources, including the Human Protein Atlas (HPA), UniProt, GTEx, and FANTOM. The goal was to determine their involvement in local or systemic secretion and to examine their spatial distribution within the human body. Each gene was assigned to a single location with the primary aim of assessing whether active secretion into the blood could be expected. Genes with experimental evidence of secretion in UniProt were kept in the secretome even without a predicted signal peptide, to include proteins secreted through non-classical pathways.

This resulted in ten different categories that can be explored in Figure 1. In summary, 785 proteins were classified as secreted to blood, with a fraction of these having other main location. Moreover 526 proteins were annotated as secreted to local compartments, e.g. male or female reproductive tissues, brain or a group of other tissues including the eye and the skin. Further, 94 proteins including salivary and pancreatic enzymes were classified as secreted to the digestive system and another 237 proteins, including laminins, collagens, elastin and fibronectin, were suggested to be involved in the forming and function of the extracellular matrix.

Additionally 625 proteins were found to be intracellular and membrane bound proteins including e.g. ER/Golgi residing proteins, mitochondrial proteins, lysosomal proteins and membrane-associated proteins. 114 genes were believed to be secreted but the final location could not be determined based on available data and they were classified as having unknown location. The last category includes the 147 predicted secreted genes encoding some of the constant, variable, joining and diversity regions of immunoglobulin genes. After excluding the genes annotated as intracellular or membrane-bound we suggest that the human secretome consists of 1903 genes having at least one secreted protein variant. More information about each secretome category, can be accessed by clicking the icons in Figure 1. Several of these genes code for multiple isoforms with alternative locations: 654 genes encode both intracellular and secreted isoforms, 134 encode both secreted and membrane-bound isoforms, and 103 encode isoforms in all three categories.

The secretome across human tissues

In most tissues, the secreted proteins are dominated by the category that matches the tissue's function (Figure 2). Proteins secreted to blood dominate the secretome of the liver, pituitary gland, parathyroid gland, choroid plexus and bone marrow, for example ALB and FGA in liver, GH1 in pituitary gland, PTH in parathyroid gland and TTR in choroid plexus. The salivary gland, stomach, pancreas and intestine are dominated by proteins secreted to the digestive system, extracellular matrix proteins make up a larger share in adipose tissue, skeletal muscle and blood vessels, and immunoglobulins in lymphoid tissues, urinary bladder and intestine.

Figure 2. The composition of the secretome across human tissues. Top: the share of each tissue's secretome expression (nTPM) contributed by each secretome category. Bottom: the number of secretome genes detected in each tissue (nTPM ≥ 1), by category. Genes classified as intracellular and membrane are not included.

The secretome across single cell types

At single cell type resolution, the secretome of specialized secretory cells is dominated by a few highly expressed genes (Figure 3). The ten most highly expressed secretome genes account for 96% of secretome expression in breast lactating cells and 91% in gastric chief cells, although these cells express 647 and 949 secretome genes, respectively. In fibroblasts, which express the broadest secretome (1,042 genes), the ten most expressed genes account for only 25%.

Single cell types also separate secretory programs that are mixed at the tissue level. In the pancreas, endocrine islet cells express proteins secreted to blood, such as insulin (INS) and glucagon (GCG), while exocrine acinar cells express digestive enzymes such as AMY2A and CPB1. In the breast, lactating cells express milk proteins such as CSN2, CSN3 and LALBA, while fibroblasts express extracellular matrix proteins such as COL6A1. In the brain, neurons express locally secreted proteins such as CBLN3, while microglia express immune proteins secreted to blood, such as CD74 and C3.

Figure 3. The composition of the secretome across selected single cell types. Top: the share of each cell type's secretome expression (nCPM) contributed by each secretome category. Bottom: the number of secretome genes detected in each cell type (nCPM ≥ 1), by category. Genes classified as intracellular and membrane are not included.

Abundance and specificity

The secretome is a rather heterogeneous group of proteins in terms of both abundance and tissue specificity. One of the most important secretory organs is the liver, which produces a large number of the proteins secreted into the blood such as albumin, fibrinogen and transferrin. Highly abundant secreted proteins also include pancreatic enzymes (PRSS1, CELA3A, AMY2A) and other digestive proteins expressed in the salivary gland (PRR4, STATH, ZG16B) or stomach (PGA3, PGA4), as well as the family of defensin proteins secreted by glandular cells in the epididymis (DEFB118, DEFB106A and DEFB129). More information on the estimated abundance in blood plasma for proteins belonging to the Secreted to blood category can be found here for immunoassay data and here for MS data.

CELA3A
CPA1
AMY1B

Figure 4. Immunohistochemistry-based images from the secreted proteins CELA3A (Chymotrypsin-like elastase family, member 3A) in pancreas, CPA1 (Carboxypeptidase 1) in pancreas and AMY1B (amylase alpha 1B) in salivary gland.

The gene expression profiles within the different secretome categories were classified based on transcriptomics analysis across all major organs and tissue types in the human body as shown in the bar plot in Figure 5. Read more about the different categories of expression here.

Prediction of transmembrane protein topology and signal peptides

Developing a better understanding of membrane protein structure and function is of immense importance for both biological and pharmacological purposes. Since membrane proteins are difficult to crystallize and severely underrepresented in structural databases, computational prediction of membrane protein structure has been crucial for continued studies of these key molecules. Most membrane protein prediction methods have focused on the topology of a-helical membrane proteins, i.e. the prediction of the position of the transmembrane (TM) segments in the protein sequence and their orientation relative to the membrane as illustrated below in the schematic view of the topology of an alpha-helical membrane protein with four transmembrane segments and extracellular N- and C-terminals.

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The TM segments are identified based on features such as length, amino acid property and hydrophobicity, and many prediction methods are based on machine-learning or deep learning techniques. Here, a selection of eight prediction algorithms was used to create a majority decision-based method (MDM), using the combined results from the chosen tools, to estimate the human membrane proteome. Each protein with at least one TM segment with overlapping predictions by four out of the eight methods is considered a membrane protein. Table 1 shows the individual results in number of predicted protein-coding genes by each method, as well as the MDM prediction.

Table 1. Prediction of the human membrane proteome by eight different prediction methods for membrane protein topology as well as the majority decision-based method MDM and a method specialized in prediction of GPCRs.

The N-terminal signal sequences that are found in most secreted proteins and some types of membrane proteins are often called signal peptides (SP). A signal peptide is primarily identified by a short hydrophobic alpha-helix combined with a number of features that enables computational prediction based on the amino acid sequence of the protein. There are also a number of methods which incorporate a SP prediction model into their TM topology prediction algorithm to enables more reliable results when it comes to distinguishing between the two features. Here the existence of signal peptides was based on a whole-proteome scan using five methods for signal peptide prediction: SignalP 6.0, Phobius, SPOCTOPUS, DeepTMHMM and DeepSig, which all have been shown to give reliable prediction results in a comparative analysis. Similarly to the MDM, a majority decision-based method for secreted proteins (MDSEC) was constructed using results from the five different prediction methods. All proteins with a predicted SP by at least three of the five methods were considered to have a signal peptide. Since signal peptides are found both in secreted proteins and in certain types of membrane proteins, proteins with a predicted SP in combination with a predicted TM region according to the MDM were considered membrane-spanning and excluded from the group of predicted secreted proteins. The remaining predicted secreted proteins were further annotated in order to exclude genes that are predicted to reside in intracellular locations such as ER or Golgi, despite having a signal peptide prediction. The resulting numbers of genes encoding a predicted secreted protein are shown in Table 2.

Table 2. Prediction of the human secretome by three different prediction methods for signal peptides as well as the MDSEC and the final prediction resulting from the annotation of the human secretome.

Classification of the human proteome

The combined results from analyses of the predicted membrane proteome and secretome were used to map the distribution of potential membrane proteins and secreted proteins in the human proteome. The protein isoforms of all human genes were annotated using the three categories: (i) secreted, (ii) membrane and (iii) intracellular (i.e., proteins with no predicted SP/TM features). Note that proteins classified as membrane may be located in intracellular membranes such as the endoplasmic reticulum or Golgi. Each of the human protein-coding genes was subsequently classified into those with all isoforms belonging to one of these groups or genes encoding protein isoforms belonging to two or all three categories (Figure 7). A gene with one membrane-spanning and one intracellular isoform, for example, is classified as intracellular and membrane. The results show that 36% of the human predicted genes have at least one protein isoform which is membrane-spanning or secreted.

Immunoglobulins and T-cell receptors

Immunoglobulin and T-cell receptor genes are included in the above categories. Of the 214 immunoglobulin genes, 139 are classified as secreted (127) or intracellular and secreted (12), most of them variable (V) gene segments. The 8 heavy chain constant genes for IgA, IgE, IgG and IgM encode both secreted and membrane-bound isoforms and are classified as intracellular, secreted and membrane, while the IgD constant gene (IGHD) is classified as membrane. The remaining 66, mainly short diversity (D) and joining (J) segments, are classified as intracellular. Of the 196 T-cell receptor genes, the 6 constant region genes are classified as membrane and the other 190, mainly variable, diversity and joining segments, as intracellular.

The secretory pathway

Most secreted proteins are secreted via the secretory pathway, as illustrated below, but interestingly some proteins including cytokines such as interleukin-1 beta (IL1B) and mitogens such as fibroblast growth factor 2 (FGF2) are secreted in a non-classical manner without entering the ER/Golgi-pathway. In the secretory pathway proteins with a signal sequence that guides them to the endoplasmic reticulum (ER) are transported from the ER through the Golgi apparatus via vesicles to arrive at the surface of the cell. The signal sequence targeting proteins for secretion, called a signal peptide, is a short, hydrophobic N-terminal sequence which is inserted into the ER membrane and subsequently cleaved off from the protein. Membrane proteins may also contain an SP, but most often the N-terminal transmembrane (TM) region function as the signal sequence. The ER signal sequences are recognized by chaperone proteins which guide the synthesizing ribosomes to the rough ER where translocation of the protein sequence occurs in a protein complex named the translocon. Membrane proteins are transferred to the lipid bilayer of the ER membrane via the translocon whereas secretory proteins are transported into the ER lumen. Once inside the ER lumen, other chaperone proteins make sure that the protein is folded and assembled correctly and the oxidative environment enables formation of disulfide bonds, addition of carbohydrates and proteolytic cleavages. The proteins that pass the ER quality control are transported via vesicles to the Golgi apparatus, where they are further modified in important processes such as glycosylation and phosphorylation. The Golgi is also responsible for sorting of proteins for transport to their final destination, which most often is the plasma membrane, lysosomes or secretion out from the cell.

Figure 8. Overview of the secretory pathway.

Relevant links and publications

Uhlén M et al., The human secretome. Sci Signal. (2019)
PubMed: 31772123 DOI: 10.1126/scisignal.aaz0274

Fagerberg L et al., Prediction of the human membrane proteome. Proteomics. (2010)
PubMed: 20175080 DOI: 10.1002/pmic.200900258

Uhlén M et al., Tissue-based map of the human proteome. Science (2015)
PubMed: 25613900 DOI: 10.1126/science.1260419