# Building Protein-Protein and Protein-Glycosaminoglycan Interaction Networks Using MatrixDB, the Extracellular Matrix Interaction Database

Coline Berthollier, Sylvain D Vallet, Madeline Deniaud, Olivier Clerc, Sylvie Ricard-Blum

► To cite this version:

Coline Berthollier, Sylvain D Vallet, Madeline Deniaud, Olivier Clerc, Sylvie Ricard-Blum. Building Protein-Protein and Protein-Glycosaminoglycan Interaction Networks Using MatrixDB, the Extracellular Matrix Interaction Database. Current Protocols, 2021, 1 (4), pp.e47. 10.1002/cpz1.47. hal-05000509

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# BUILDING PROTEIN-PROTEIN AND PROTEIN-GLYCOSAMINOGLYCAN INTERACTION NETWORKS USING MATRIXDB, THE EXTRACELLULAR MATRIX INTERACTION DATABASE

Coline Berthollier,¹ Sylvain D. Vallet,¹ Madeline Deniaud,¹ Olivier Clerc,¹ and Sylvie Ricard-Blum¹,²

¹Univ Lyon, University Claude Bernard Lyon 1, CNRS, INSA Lyon, CPE, Institute of Molecular and Supramolecular Chemistry and Biochemistry, UMR 5246, F-69622 Villeurbanne cedex, France ;

²Corresponding author: sylvie.ricard-blum@univ-lyon1.fr, Phone number: +33(0)472448232

## SIGNIFICANCE STATEMENT:

Matrix Database (MatrixDB) focuses on extracellular matrix (ECM) and on experimentally-supported protein-protein and protein-glycosaminoglycan interactions. MatrixDB also provides a user-friendly interface for building specific interaction networks online, using advanced queries, expression and quantitative proteomics data. This article reports detailed protocols to browse MatrixDB content, to use the Advanced search mode to create lists of biomolecules based on user criteria and their interaction networks, to build tissue-specific interaction networks, and to visualize and analyze them. The last protocol focuses on the building of tridimensional model of glycosaminoglycan oligosaccharides binding to proteins using the GAG Builder tool hosted by MatrixDB.

## ABSTRACT:

The interaction database MatrixDB (http://matrixdb.univ-lyon1.fr/) reports protein-protein and protein-glycosaminoglycan interactions in human, mammalian and model organisms, involving at least one extracellular matrix (ECM) constituent, namely full-length proteins, ECM multimeric proteins considered as stable complexes, proteoglycans, glycosaminoglycans (GAG) and bioactive fragments called matricryptins, which are released upon limited proteolysis of ECM proteins. The current version of MatrixDB (as of October 2020) contains 106 543 experimentally-supported interactions, any type of biomolecule combined. MatrixDB is the only database focusing on the curation of ECM protein and GAG interactions. The iNavigator integrated in MatrixDB allows users to build interaction networks online and to filter them according to expression data, quantitative proteomics data or interaction detection methods. MatrixDB belongs to the International Molecular Exchange (IMEx) consortium and uses its curation rules to capture interaction data, which are available in standardized exchange formats according to the Human Proteome Organization - Proteomics Standards Initiative (HUPO-PSI) standards.

**Basic Protocol 1:** Browse MatrixDB

**Basic Protocol 2:** Create a list of biomolecules of interest to build interaction networks

**Basic Protocol 3:** Build and export interaction networks of selected biomolecules using the iNavigator

**Basic Protocol 4:** Build specific interaction networks using the iNavigator widgets
**Basic Protocol 5:** Generate 3D models of glycosaminoglycan oligosaccharides using the GAG Builder tool

**KEYWORDS:** interaction database • extracellular matrix • interaction network • protein-glycosaminoglycan interactions • protein-protein interactions

## INTRODUCTION:

MatrixDB is a publicly available interaction database (http://matrixdb.univ-lyon1.fr/) focused on the extracellular matrix, reporting protein-protein and protein-glycosaminoglycan (GAG) interactions (Chautard et al., 2011, Chautard et al., 2009). In addition, MatrixDB allows users to build interaction networks using its integrated iNavigator (Launay et al., 2015) and to filter them in order to get specific networks based on transcriptomic and proteomic data, and the combination of several criteria (Clerc et al., 2019a). MatrixDB is a member of the International Molecular Exchange (IMEx) consortium (https://www.imexconsortium.org/) (Orchard et al., 2012; Porras et al., 2020), and MatrixDB is one of the services of ELIXIR-FR (https://www.france-bioinformatique.fr/elixir-fr/), the French node of ELIXIR (European Life-science Infrastructure for Biological Information) since 2019.

Interaction data stored in MatrixDB are experimentally-supported and manually curated from the literature using the IMEx curation rules and the IntAct curation interface (Orchard et al., 2014), and also include ECM-related interaction data imported from other IMEx interaction databases (Chautard et al., 2011, Launay et al., 2015). Furthermore, MatrixDB has implemented the PSICQUIC tool (Proteomics Standards Initiative Common Query Interface, Aranda et al., 2011) to query multiple interaction databases simultaneously. MatrixDB data are freely and publicly available and can be downloaded under the MI-TAB 2.7 standard formats defined by HUPO-PSI (Human Proteome Organization – Proteomics Standards Initiative, http://www.psidev.info/). They are distributed under the terms of the Creative Commons Attribution License CC BY 4.0 according to the FAIR (Findability, Accessibility, Interoperability and Reusability) guiding principles (Wilkinson et al., 2016).

Here we provide users with protocols to browse MatrixDB content, to build networks of a single biomolecule or a list of biomolecules, and specific networks of a tissue, based on expression or quantitative proteomic data, a disease using OMIM data (Amberger & Hamosh, 2017), a molecular function or a biological process based on UniProtKB keywords (The UniProt Consortium, 2019) or Gene Ontology (GO) terms (The Gene Ontology Consortium, 2019), an interaction detection method or any combination of these criteria. MatrixDB being the only database focusing on glycosaminoglycan interactions, we also give a protocol to generate tridimensional models of glycosaminoglycan sequences binding to proteins using the GAG Builder tool implemented in MatrixDB (Clerc et al., 2019a, Clerc et al., 2019b).

## BASIC PROTOCOL 1: BROWSE MATRIXDB

This protocol shows how to access report pages of biomolecules, associations and experiments and which data can be retrieved from them.
**Materials:**

**Hardware:** Computer with Internet connection

**Software:** Web browser such as Chrome (recommended) or Mozilla Firefox

## Querying MatrixDB using the homepage search bar

1. Go to MatrixDB homepage (http://matrixdb.univ-lyon1.fr/), enter a query in the search bar as shown in **Figure 1** to display the biomolecules, UniProtKB keywords or publications related to the query, ranked by the number of interactions for biomolecules and publications, and by alphabetical order for UniProtKB keywords. Queries can be made to search for biomolecules using their common names, MatrixDB identifiers, UniProtKB accession numbers for proteins, EBI Complex-Portal identifiers for multimers considered as stable complexes (Meldal et al., 2019) and ChEBI identifiers for small molecules and glycosaminoglycans (Hastings et al., 2016) with a cross-reference to GlyTouCan (Tiemeyer et al., 2017) when available. UniProtKB keywords and publications containing the query in their name or definition for keywords or in their title or abstract for publications are retrieved too. Publications can also be searched using author names or PubMed Identifiers. For example, "Collagen I" as a query returns 164 biomolecules, including human collagen I, which has 30 interactions in MatrixDB, 7 UniProtKB keywords and 160 publications.

*Note: When using the homepage search bar UniProtKB keywords but not Gene Ontology (GO) terms can be used as queries. Only the Advanced search mode (see Basic protocol 2) can be used to query MatrixDB with GO terms.*

2. Tick the "human only" box in the "Biomolecules" category to select human biomolecules of Collagen I. This returns 91 biomolecules, 7 UniProtKB keywords and 160 publications as shown in **Figure 1**. Pass the cursor over Human collagen I to open a pop-up window displaying MatrixDB identifier, common name of the biomolecule and its species (e.g., "MULT_3_human", "collagen-I", and "Human").

*Note: For monomeric proteins, UniProtKB accession numbers are displayed.*

## View the Biomolecule report page

3. Click on "Collagen-I" entry to display the Biomolecule report page. "General data and interactions" is highlighted in the black horizontal menu at the top of the page, where are displayed information on the biomolecule on the left of the screen and the list of its partners on the right (**Figure 2**).

4. Collagen I, being an heterotrimer, its stoichiometry and composition are indicated using UniProtKB accession numbers: two α1 chains (P02452) and one α2 chain (P08123). The cross-reference with EBI Complex Portal identifier (CPX-1650) is provided.
Note: UniProtKB keywords and Gene Ontology (GO) terms are listed on the Biomolecule report page when available as shown in **Figure 3** for the α1 chain of collagen I (P02452).

Human collagen I has 34 partners in MatrixDB, 18 partners identified in 24 experiments using human collagen I (in green) and 16 partners inferred by homology (in orange) from experiments performed with non-human collagen I (Figure 2). Partners and species can be sorted by alphabetical order, and the number of experiments can be ranked in increasing number of supporting experiments.

Note: The PSICQUIC tool (Aranda et al., 2011) implemented in MatrixDB can be used to retrieve additional interaction data from other databases, including those of the IMEx consortium.

The partners can be selected by entering a term in the Filter box on the top of the partner list. Entering "bone" in this field returns 3 partners containing bone in their names (human and murine bone morphogenetic protein 1 and human bone sialoprotein 2).

5. In the black horizontal menu at the top of the biomolecule report, click on "Expression and Proteomics" to display expression data (Figure 2) provided by Gene Expression Atlas and obtained by RNA-sequencing in 53 human tissue samples from the Genotype-Tissue Expression (GTEx) Project (The GTEx Consortium et al., 2015). Expression data are visualized as heat maps of the number of transcripts per million (TPM) in tissues. A pop-up window opens when passing the cursor over the boxes to display the gene name, the organism part, the expression level in TPM, and the number of biological replicates. With human collagen I being a heterotrimer, the expression data of both genes (COL1A1 and COL1A2) encoding its constitutive polypeptide chains are shown.

Note: Quantitative proteomic data are displayed below expression data when available. MatrixDB contains 14 quantitative proteomic datasets (Clerc et al., 2019a) from normal and tumor basement membranes (http://matrisomeproject.mit.edu/ecm-atlas, Naba et al., 2016).

6. Click on "Download" to display and save expression data as a .tsv file.

**View the Association report page**

7. In the interaction list displayed in step 4 (Figure 2), click on the number of supporting experiments for "heparin" (GAG_1) to display the association name (GAG_1_MULTI_3_human) and the list of supporting experiments performed with human collagen I (MULT_3_human, green) and with non-human collagen I (orange).

To access the Association report page, listing all the experiments, click on "GAG_1_MULTI_3_human". Alternatively click on a specific experiment (e.g., GAG_1_MULTI_3_human_2524477_MatrixDB_1) to access directly the Experiment report page of this interaction.
Note: MatrixDB association identifiers comprise both MatrixDB identifiers of both partners in alphanumerical order. The PMID of the publication reporting the experiment, the name of the source database, and a number incremented according to the number of experiments supporting the association are added to the association name to form MatrixDB identifiers of experiments. All items are separated by underscores.

## View the Experiment report page

8. In the heparin-human collagen I association report, click in the blue panel on the first supporting experiment (GAG_1__MULT_3_human_2524477_MatrixDB_1) to access the experiment report, which includes the interaction and participant detection methods, the interaction type (direct interaction or physical association), the experimental role (bait or prey) of participants, the PMID of the publication reporting the experiment, and when available the binding site, kinetic parameters and affinity, stoichiometry, and the effect of mutations (Figure 4).

## View the Publication report

9. Click on PMID 2524477 to access the publication report (Figure 5), which comprises the title, authors, journal, publication date, abstract, a link to PubMed, and a table listing the experimentally supported interactions reported in the publication.

10. Click on MatrixDB logo on the top left of the screen to go back to MatrixDB home page.

## BASIC PROTOCOL 2: CREATE A LIST OF BIOMOLECULES OF INTEREST TO BUILD INTERACTION NETWORKS

In addition to the search bar available on the home page, MatrixDB has an Advanced search mode. A list of biomolecules of interest can be built according to combination of criteria set by the users, and then used to build interaction networks.

## Materials:

See Basic Protocol 1.

## The Advanced search mode

1. There are three ways to access the Advanced search from MatrixDB home page (see Basic Protocol 1 and Figure 1):

- Click on "Advanced search" below the search bar on the home page

- OR Click on "Build interaction network" logo at the top right of the home page

- OR Click on "Build interaction networks" on the left, and then click on "Advanced query".
Note: The displayed page contains a quick user guide.

Four options are available on the left of the screen to select biomolecules:

- Biomolecule information (free text)

- UniProtKB keywords & GO terms (controlled vocabulary)

- Publications

- Diseases (free text, UniProtKB keywords or Online Mendelian Inheritance in Man - OMIM - annotations (Amberger & Hamosh, 2017)).

Two examples of biomolecule selection are detailed below using a combination of functional annotations or diseases.

## Build a biomolecule list combining UniProtKB keywords and GO terms

Note: UniProtKB (The UniProt Consortium, 2019) and Gene Ontology (The Gene Ontology Consortium, 2019) provide annotations on the structure, function and location of proteins and gene products.

2. Select "Keywords & GO terms" in the menu on the left of the screen.

3. Enter the UniProtKB keyword "Collagen degradation" (or its identifier "KW-0177") and the GO term "Collagen catabolic process" (or its identifier "GO:0030574") separated by a comma (Figure 6).

Note: Space-separated words are considered as a single request, whereas comma-separated queries are considered as multiple requests.

4. Select "At least one term needed", "Human biomolecules only" and/or "Biomolecules involved in at least one interaction" before clicking on "Show list". Four primary hits are returned including the annotation source, the identifier, the name and number of the biomolecules annotated (Figure 7). There is no secondary hit.

Note: The results of advanced queries are sorted by primary and secondary hits. When the query is found in the name/identifier of the biomolecule or in the title of the publication, the results are considered as primary hits. When it is found in the biomolecule description or in the publication abstract, the results are considered as secondary hits.

5. Click on "Show list of biomolecules" to display the name, identifier, species and the number of partners of the listed biomolecules list of biomolecules.

6. Click on "Back to hits" at the top right of the screen to go back to the hits.

7. Click on the "+13" and "+30" buttons to add "collagen degradation" and "collagen catabolic process"-related biomolecules to the cart (Figure 7).
Note: The biomolecule list is available in the table located at the right of the screen, including the common name and number of partners of biomolecules. Additional options above the table allow to show previous queries, and to reload or empty the cart. All the listed biomolecules can be added in a single step by clicking on "Add all biomolecules to cart" below the table of primary hits. Users can manually check the biomolecule list and delete unwanted entries.

Users can add further biomolecules to the list by clicking on "New query" on the top right of the screen to make a new search.

Genetic diseases-related biomolecules may be retrieved from MatrixDB using OMIM annotations or UniProtKB keywords. Searching for epidermolysis bullosa dystrophica using the OMIM identifier 226600 leads to one primary hit related to collagen α1(VII) chain (Figure 8A), whereas searching for "Epidermolysis bullosa dystrophica" returns 5 primary hits and 1 secondary hit (Figure 8B), all also containing the collagen α1(VII) chain.

8. When the list of biomolecules is set, click on "Build interaction network" to open the iNavigator page (see Basic Protocol 3).

**BASIC PROTOCOL 3: BUILD AND EXPORT NETWORKS OF SELECTED BIOMOLECULES IN THE iNAVIGATOR**

This protocol aims to demonstrate how to use the cart for collecting biomolecules of interest, and then how to build and save their interaction network with the iNavigator.

**Materials:**

See Basic Protocol 1.

**Use the cart to store biomolecule(s), annotation(s) and/or publication(s) of interest**

1. Using the MatrixDB home page search bar, go to the human collagen I Biomolecule report page (see Basic Protocol 1, steps 1-4).

2. Click on the cart icon located in the left box (named Multimeric complex) to put the selected biomolecule into the cart for later use. It triggers the flashing of the network icon located at the right of the cart.

Note: Biomolecules, UniProtKB keywords, GO terms and publications (PMIDs) can be added to the cart by clicking on the cart icon. Click on the cart in the black horizontal menu at the top of the screen to see its content. Unwanted entries can be removed from the cart.
## Build interaction networks with the iNavigator

3. Click on the network icon in the black horizontal menu located at the top of the screen, then on "Build items interactions network" to build the human collagen I interaction network (Figure 9).

Note: During data loading to create the network, interactions represented by edges are displayed as dashed lines, and as lines when loading is complete. The iNavigator selects the network view, which minimizes the overlapping of nodes (biomolecules). Several layouts might fulfill this criterion and the visualization of the same interaction network may vary.

Passing the cursor over a node to show the common name of the biomolecule, the gene name (for polypeptide chains) and the species. A table on the right of the screen keeps track of examined molecules and edges.

4. Click and drag nodes to move them individually and organize the network.

5. Click on an edge to display information on the interaction, with a link to the experiment report.

6. Use directional arrows on the upper left of the screen to move the whole network. Use autofocus to optimize network visualization, and plus and minus to adjust its size.

Note: Further biomolecules, annotations or publications could be added to a network using the Box widget of the iNavigator (see Basic Protocol 4)

## Save networks for re-use in the iNavigator, export to Cytoscape or a publication

7. MatrixDB iNavigator provides three options available by clicking on "Save button" on the top right of the iNavigator (Figure 9):

- Take a snapshot: export the network as a vector image in .svg format (no legend displayed).

- Export to Cytoscape: download a zipped folder containing two files, the simple interaction (.sif) and the node attribute (.na) files, for further analysis in Cytoscape, an open source software platform for visualizing complex networks (Shannon, 2003).

- Save network: a personal key is attributed to the network, which is available for one month after the creation of the network. Use the personal key to reload the network in the iNavigator.

## BASIC PROTOCOL 4: BUILD SPECIFIC INTERACTION NETWORKS USING THE iNAVIGATOR WIDGETS

This protocol aims to demonstrate how to build specific interaction networks using filters and the four widgets of the iNavigator:
- Tabular network widget

- Box widget

- Filter network widget

- Palette widget

The widgets open independently and are closed by clicking on the minus sign in the upper left corner.

**Materials:**

See Basic Protocol 1 for hardware and software

To access the iNavigator as shown in **Figure 9**, users must build a new network or reload a previously built network using the personal key allocated upon its creation (see Basic Protocol 3, step 7). This protocol is described using the collagen I network built with Basic Protocol 3.

**Use the Tabular network widget**

1. Select the first widget on the left to display the table containing the names and identifiers of the biomolecules present in the network, and their interactions including the interaction type ("genuine" or "inferred by homology") and the number of supporting experiments (Figure 10).

2. Click on "Toggle node labels" in the table to display the name and species of each node. Labels are hidden by default.

3. Click on the biomolecule identifier in the "Biomolecules" tab of the table to go back to the Biomolecule report page (see Basic Protocol 1).

4. Select nodes in the network by ticking or unticking boxes on the left to hide the selected nodes, highlight them in the network with a blue contour, or add them to the cart for building further networks. Human biomolecules can be selected simultaneously as shown in **Figure 10**, and the selection can be inverted.

5. Click on the number of interactions in the table to highlight the biomolecules involved in these interactions with a light blue line in the displayed network.

6. Click on the number of experiments in the "Interactions" tab to go back to the Association report page.

7. When done, close the widget by clicking on the minus sign.

**Add further biomolecules to a network using the Box widget**

8. In the iNavigator, select the Box widget on the left (Figure 11).
9. Enter the biomolecule name or free text in the search bar of the iNavigator to search for additional biomolecules and their partners either in MatrixDB or in the network currently displayed. "Collagen II" returns 42 biomolecules in the first column with human collagen II in the 6th row, and 59 publications.

Note: Querying MatrixDB as shown in the example is useful to add to the network further biomolecules of interest and their partners. Querying the current network is useful to add all partners of a biomolecule present in the network.

10. Tick the box on the left of "Collagen II", which adds it to the widget box where its identifier (MULT_9_human) is displayed. Click on "Collagen II" common name to go back to the Biomolecule report page.

Note: It is also possible to select a biomolecule in the network and to add it into the Box widget by double left-clicking on the biomolecule node and then by clicking on the green plus sign that appears in the upper right corner of the Box widget.

11. Click on "get interactome" in the Box widget to add collagen II and its partners to the current network. The Tabular network widget is updated accordingly.

12. The Box widget can be emptied by clicking on "Clear", but this does not delete added nodes and edges in the network.

Note: The addition of biomolecules to the network is irreversible at this stage. Users must save networks prior to new queries and additions.

**Build specific interaction networks using the Filter network widget**

This widget allows users to reversibly filter the interactions of the displayed network using three filters:

- Expression data

- Quantitative proteomic data

- Interaction detection methods

Note: Data can be sorted by the number of biomolecules of the network expressed (expression data) or present at the protein level (quantitative proteomic data) in each tissue or by number of experiments for the interaction detection methods. It is possible to set a threshold in transcripts per million (TPM) for expression data and in arbitrary units (AU) for quantitative proteomic data.

**Build tissue-specific interaction networks based on expression data:**

Tissues are listed by alphabetical order by default (Figure 12). The sub-network of human collagens I and II in the aorta can be built as follows:

13. In the Filter network widget, select "Gene Expression" and "Aorta" (20 biomolecules, **Figure 12**). The nodes are color-coded according to their transcriptomic data:
- Cyan: no expression data available

- Green: gene expressed (above the threshold when set) in the selected tissue.

- Red: gene not expressed, or expressed below the threshold level, in the selected tissue. When clicking on "Apply", these nodes are no longer displayed in the network.

Note: Expression data are not available for multimers, glycosaminoglycans and small molecules, which are thus automatically deleted from the network.

14. Set the threshold to 50 transcripts per million to display only proteins expressed above this threshold in aorta.

15. Untick the "aorta" dataset to go back to the initial network.

**Build tissue-specific networks based on quantitative proteomic data:**

The table contains datasets for isolated polypeptide chains in human (h) and murine (m) tissues, and healthy or tumor tissues, mostly basement membranes (Figure 13). Proteins present in tissues at levels above the threshold defined by users are in green, and those present in tissues below the threshold level are masked.

16. Select "Quantitative proteomics", tick the box corresponding to the human primary metastatic colorectal cancer dataset (h. Prim. Me. Colorect. Ca), set a 100 000 A.U. threshold and click on "Apply" to get the network shown in Figure 13.

Note: **Figure 14** gives another example of the building of a tissue-specific interaction network. The network built using ECM assembly as a query (Figure 14A) is filtered using quantitative proteomic data collected in colorectal cancer-derived liver metastasis tissue (Figure 14B).

**Build specific interaction networks based on interaction detection methods:**

Filtering interactions by their detection method might be useful to discriminate direct interactions from physical associations or to select methods allowing the calculation of kinetic parameters and affinity. Interaction detection methods can be sorted according to the number of experiments identified as shown in Figure 15.

17. Select "Interaction detection method".

18. Use the arrow to sort the "Number of experiments" column by descending order. In the example, the largest number of experiments were performed by surface plasmon resonance (SPR) arrays.

19. Tick the "surface plasmon resonance array" box and click on "Apply" to display in green the 87 associations supported by experiments performed with this method.
Customize the visualization of the network using the color palette

The fourth widget is a color palette for user-driven layout customization (Figure 16). Users can choose among a wide color spectrum to color a specific subset of selected nodes.

20. Use the selection option of the first widget to select human nodes. Human nodes are highlighted with a light blue thick outline (see Basic Protocol 4, step 4) even after the first widget has been closed.

21. Open the color palette and select a color (e.g., yellow as shown in Figure 16).

BASIC PROTOCOL 5: GENERATE 3D MODELS OF GLYCOSAMINOGLYCAN OLIGOSACCHARIDES USING THE GAG BUILDER TOOL

The GAG builder tool, a version of Polys Glycan Builder (Engelsen et al., 2014) dedicated to GAGs has been developed and implemented in MatrixDB to build tridimensional models of GAG oligosaccharides binding to proteins. The 3D models of GAGs are generated from the GlycoCT codes available on the Biomolecule report page via a CT23D converter (Clerc et al., 2019b) and visualized with LiteMol (Sehnal et al., 2017). The protocol detailed below explains how to find the GlycoCT code in MatrixDB, and to convert it into a tridimensional model using GAG Builder and LiteMol viewer. The GlycoCT code describes monosaccharide residues (RES), linkages between monosaccharides (LIN), alternative configurations (ALT, iduronic acid or glucuronic acid in heparin, heparan sulfate and dermatan sulfate), chemical groups, and the number of disaccharide repeats (REP) (Herget et al., 2008).

Materials:

See Basic Protocol 1

Use the GAG Builder tool to generate 3D models of GAG oligosaccharides

1. Search for "chondroitin sulfate A" using the homepage search bar on the MatrixDB main page (see Basic Protocol 1). Chondroitin sulfate A appears in the "Biomolecules" category and has 10 partners in MatrixDB.

2. Click on the chondroitin sulfate A entry to access its biomolecule report (Figure 17). Chondroitin sulfate A disaccharide is represented using the Symbol Nomenclature For Glycans (SNFG).

3. To get its GlycoCT code, click on "Get code" in the GlycoCT code box. Select the entire code, copy it and hide it.

4. Go back to the MatrixDB main page by clicking on the logo at the top left of the screen.
5. Click on "GAG Builder" on the "Tools" menu.

6. Paste the GlycoCT code in the left panel and click on "Submit" to visualize the 3D model in the LiteMol viewer in the right panel.

Note: Ambiguous GlycoCT codes are not supported by GAG builder and therefore lead to the following error message: "Your input doesn't look like a valid GlycoCT code". To avoid it, the uronic acid residue features within the disaccharide pattern of the glycosaminoglycan must be well-characterized. Detailing several alternative residues does not resolve the problem, because the GAG Builder tool is not able to take them into account. It explains why heparin, heparan sulfate and dermatan sulfate GlycoCT codes provided in MatrixDB are not recognized and thus cannot be converted into tridimensional models. In addition, unsaturated residues generated upon enzymatic digestion of glycosaminoglycans are not incorporated in the GlycoCT format.

7. In the LiteMol viewer, zoom in and out on the 3D model by moving the cursor back and forth while pressing the right button of the mouse (Figure 18). Four options to customize the view of the model are available in the upper part of the box where the model is displayed ("Scene options", "Show controls", "Expand" and "Reset scene"). A fifth option, "Screenshot", can be used to save the model as an image.

8. Save the atomic coordinates of the 3D model as a .pdb file by clicking on "Download File" for later use in another 3D molecular viewer.

9. Click on "New conversion" in the upper part of the screen to go back to MatrixDB home page and make a new query to build the 3D model of another GAG sequence.

COMMENTARY

**BACKGROUND INFORMATION:** MatrixDB (http://matrixdb.univ-lyon1.fr/) is an interaction database focused on the extracellular matrix. It contains experimentally-supported and manually-curated protein-protein and protein-glycosaminoglycan interactions. MatrixDB belongs to the International Molecular Exchange (IMEx) consortium, and follows its curation rules to capture interaction data, which are available in standardized exchange formats according to the Human Proteome Organization - Proteomics Standards Initiative (HUPO-PSI) standards. The iNavigator integrated in MatrixDB allows users to build interaction networks online, and to filter them according several criteria (e.g., expression data, quantitative proteomic data, disease, molecular function, biological process or interaction detection methods). MatrixDB is distributed under the terms of the Creative Commons Attribution License CC BY 4.0, and is one of the services of ELIXIR-FR (https://www.france-bioinformatique.fr/elixir-fr/), the French node of ELIXIR (European Life-science Infrastructure for Biological Information) since 2019.
**UNDERSTANDING RESULTS:** Four protocols to build global and specific interaction networks of MatrixDB, and a protocol to generate three-dimensional models of GAG sequences binding to proteins are provided here. They cover the major applications of MatrixDB. The Advanced search query allows users to browse MatrixDB in order to build a list of biomolecules of interest, and then to build and visualize tissue-specific and disease-specific interaction networks based on transcriptomic and quantitative proteomic data, and networks associated with a molecular function and/or a biological process. These contextualized networks are useful to decipher the molecular mechanisms underlying physiological and pathological processes. Networks generated in MatrixDB can be exported to the open source software platform Cytoscape (https://cytoscape.org/) to be visualized using different layouts, and analyzed by Cytoscape Apps. Furthermore, the GAG Builder tool integrated into MatrixDB may be used to build 3D models of glycosaminoglycan sequences binding to proteins.

**CRITICAL PARAMETERS AND TROUBLESHOOTING:**

* The standard Search bar is used to query MatrixDB based on free text and UniProtKB keyword identifiers but not on GO term identifiers.

* The Advanced search mode must be used to query MatrixDB with GO term identifiers. Data added to a network using the iNavigator box widget cannot be deleted from the network. The users must thus save the network before adding further biomolecules in order to be able to reuse it.

* Ambiguous GlycoCT codes (e.g., uronic acid instead of glucuronic acid or uronic acid) are not supported by GAG Builder.

**TIME CONSIDERATIONS:** The basic protocols can be individually completed within 10 min. It might take longer for basic protocols 2 and 3, which both require data loading to build networks, depending on the number of biomolecules included in the networks. Extra time will be also needed to perform multiple queries using the Advanced search mode, and to generate specific networks using iNavigator filters.

**ACKNOWLEDGEMENTS:**

We thank Nicolas Thierry-Mieg (TIMC-IMAG, UMR 5525, Université Grenoble Alpes, France) for his constant support throughout the creation and the development of MatrixDB. MatrixDB was supported by Fondation pour la Recherche Médicale (DBI20141231336 to MD and SRB), Institut Français de Bioinformatique (ANR-11-INBS-0013, Glycomatrix project, call 2015 to OC and SRB), and by CNRS (GDR 3739, Structure, Fonction et Régulation des Glycosaminoglycanes to SRB).

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Chautard, Emilie, Ballut, L., Thierry-Mieg, N., & Ricard-Blum, S. (2009). MatrixDB, a database focused on extracellular protein-protein and protein-carbohydrate interactions. *Bioinformatics*, 25(5), 690-691. https://doi.org/10.1093/bioinformatics/btp025

Clerc, O., Deniaud, M., Vallet, S. D., Naba, A., Rivet, A., Perez, S., Thierry-Mieg, N., & Ricard-Blum, S. (2019a). MatrixDB : Integration of new data with a focus on glycosaminoglycan interactions. *Nucleic Acids Research*, 47(D1), D376-D381. https://doi.org/10.1093/nar/gky1035

Clerc, O., Mariethoz, J., Rivet, A., Lisacek, F., Pérez, S., & Ricard-Blum, S. (2019b). A pipeline to translate glycosaminoglycan sequences into 3D models. Application to the exploration of glycosaminoglycan conformational space. *Glycobiology*, 29(1), 36-44. https://doi.org/10.1093/glycob/cwy084

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## KEY REFERENCES:

Chautard, E., Ballut, L., Thierry-Mieg, N., & Ricard-Blum, S. (2009). MatrixDB, a database focused on extracellular protein-protein and protein-carbohydrate interactions. *Bioinformatics*, 25(5), 690-691.
https://doi.org/10.1093/bioinformatics/btp025

*The first description of MatrixDB as a database focused on extracellular matrix reporting mammalian protein-protein and protein-carbohydrate interactions.*

Chautard, E., Fatoux-Ardore, M., Ballut, L., Thierry-Mieg, N., & Ricard-Blum, S. (2011). MatrixDB, the extracellular matrix interaction database. *Nucleic Acids Research*, 39 part 2, D235-D240.
https://doi.org/10.1093/nar/gkq830

*An in-depth description of MatrixDB architecture, content and specific features associated with the extracellular matrix (ECM), and its adoption of the HUPO-PSI and IMEx standards for the curation and exchange of interaction data.*

Launay, G., Salza, R., Multedo, D., Thierry-Mieg, N., & Ricard-Blum, S. (2015). MatrixDB, the extracellular matrix interaction database : Updated content, a new navigator and expanded functionalities. *Nucleic Acids Research*, 43(D1), D321-D327. https://doi.org/10.1093/nar/gku1091

*This update of MatrixDB includes the creation of a new website and the development of a graphical navigator, the iNavigator, to build, filter (e.g., using expression data or interaction methods), expand and save interaction networks.*
Clerc, O., Deniaud, M., Vallet, S. D., Naba, A., Rivet, A., Perez, S., Thierry-Mieg, N., & Ricard-Blum, S. (2019). MatrixDB: Integration of new data with a focus on glycosaminoglycan interactions. *Nucleic Acids Research*, 47(D1), D376-D381. https://doi.org/10.1093/nar/gky1035

*Cross-referencing of GAG sequences with the major glycan repository GlyTouCan, their visualization under various formats (IUPAC, GlycoCT and SNFG) and the building of their 3D models with the GAG Builder tool were implemented in MatrixDB. Quantitative ECM proteomic datasets, and an "Advanced search" mode were added to browse MatrixDB content with combined criteria.*

## FIGURE LEGENDS:

**Figure 1. MatrixDB home page.** Items used in the protocols are highlighted with colored outlines: red for home page Search bar and results, green for options to go to "Advanced search", orange for the GAG Builder tool and purple for MatrixDB logo to go back to MatrixDB home page. Results for the "collagen I" query are displayed in 3 categories. Only human biomolecules are shown.

**Figure 2. The human collagen I Biomolecule report page.** It displays information on this heterotrimeric biomolecule, its interactions and expression data.

**Figure 3. The human collagen α1(I) chain Biomolecule report page.** Additional data are available compared to the human collagen I entry, including protein domains, biological function(s) and location (red), UniProtKB keywords (green) and Gene Ontology (GO) terms (orange).

**Figure 4. Experiment report page for human collagen I - heparin interaction.**

**Figure 5. Publication report page.** PMID 2524477 reports 9 interactions, including collagen I - heparin interaction.

**Figure 6. Advanced search using UniProtKB keywords and GO terms - Queries.** Combined advanced search with the UniProtKB keyword "Collagen degradation" and the GO term "Collagen catabolic process", restricted to human biomolecules involved in at least one interaction, but extended to all biomolecules annotated by at least one of these two annotations.

**Figure 7. Advanced search using UniProtKB keywords and GO terms - Results.** Four primary hits match the query "Collagen degradation" or "Collagen catabolic process".

**Figure 8. Advanced search for diseases. (A)** Results using "226600", the *epidermolysis bullosa dystrophica* OMIM identifier returning one primary hit. **(B)** Results using the disease name *epidermolysis bullosa dystrophica* as a query returning five primary hits and one secondary hit. Queries were restricted to human biomolecules involved in at least one interaction in both cases.

**Figure 9. Human collagen I interaction network built in the iNavigator.** The iNavigator contains a legend (top-right), a tracking table (bottom-right), directional arrows and zoom options (top-left). Options to save built networks are shown in a red-contour box (top-right). In the network, nodes are
color- and shape-coded as a function of the biomolecule type: blue squares for proteins, orange diamonds for multimers, green circles for glycosaminoglycans, cyan crosses for bioactive fragments and red, yellow and magenta triangles for cations, lipids and small molecules (including synthetic peptides) respectively.

**Figure 10.** The Tabular network widget of the iNavigator and its selection options (left). In the human collagen I network (right), human biomolecules are highlighted with a light blue thick outline.

**Figure 11.** The Box widget of the iNavigator and the addition of biomolecules and interactions to a network. The iNavigator search bar (top-left, red) was used to search for human collagen II, which was first added to the box widget (bottom-left), and then added to the network (right).

**Figure 12.** A tissue-specific network of human collagens I and II in aorta, using expression data and a 50-TPM threshold. Green nodes: genes expressed above 50 TPM, cyan nodes: no expression data available.

**Figure 13.** A tissue-specific network of human collagens I and II in human primary metastatic colorectal cancer tissue, using quantitative proteomic data and a 100 000-A.U. threshold. Green nodes: proteins present in higher amount than 100 000 A.U., cyan nodes: no quantitative proteomic data available.

**Figure 14.** The global network and a tissue-specific network of human biomolecules involved in extracellular matrix assembly built in the iNavigator. (A) The global network was built using the advanced search mode and the GO term "Extracellular matrix assembly", restricted to human biomolecules involved in at least one interaction. (B) A tissue-specific network was obtained by filtering the global network using quantitative proteomics data collected in human colorectal cancer-derived liver metastasis tissue.

**Figure 15.** A sub-network of human collagens I and II identified by surface plasmon resonance (SPR) array.

**Figure 16.** The Palette widget of the iNavigator. Human biomolecules within the human collagens I and II network were selected using the Tabular network widget (not shown) and colored in yellow using the Palette widget.

**Figure 17.** A glycosaminoglycan Biomolecule report page. The Symbol Nomenclature For Glycans (SNFG) representation of the disaccharide and the GlycoCT code (red) of chondroitin sulfate A are displayed.

**Figure 18.** Building a three-dimensional model of a GAG sequence. Chondroitin sulfate A GlycoCT code (left panel) and tridimensional model visualized with LiteMol (right panel), obtained with the CT23D converter we developed (Clerc *et al.*, 2019b).
<figure><img src="image_3.png" /><figcaption>MatrixDB home page</figcaption></figure>

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<figure><img src="image_4.png" /><figcaption>The human collagen I Biomolecule report page</figcaption></figure>

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<figure><img src="image_5.png" /><figcaption>The human collagen α1(I) chain Biomolecule report page</figcaption></figure>

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<figure><img src="image_6.png" /></figure>

Experiment report page for human collagen I - heparin interaction

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<figure><img src="image_7.png" /></figure>

Publication report page.

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<figure><img src="image_8.png" /></figure>

Advanced search using UniProtKB keywords and GO terms - Queries

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<figure><img src="image_9.png" /></figure>

Advanced search using UniProtKB keywords and GO terms - Results

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<figure><img src="image_10.png" /></figure>

Advanced search for diseases

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<figure><img src="image_11.png" /><figcaption>Human collagen I interaction network built in the iNavigator.</figcaption></figure>

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<figure><img src="image_12.png" /><figcaption>The Tabular network widget of the iNavigator and its selection options (left)</figcaption></figure>

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<figure><img src="image_13.png" /><figcaption>The Box widget of the iNavigator and the addition of biomolecules and interactions to a network
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<figure><img src="image_14.png" /></figure>

A tissue-specific network of human collagens I and II in aorta, using expression data and a 50-TPM threshold

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<figure><img src="image_15.png" /></figure>

A tissue-specific network of human collagens I and II in human primary metastatic colorectal cancer tissue, using quantitative proteomic data and a 100 000-A.U. threshold

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<figure><img src="image_16.png" /></figure>

The global network and a tissue-specific network of human biomolecules involved in extracellular matrix assembly built in the iNavigator

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<figure><img src="image_17.png" /><figcaption>A sub-network of human collagens I and II identified by surface plasmon resonance (SPR) array.</figcaption></figure>

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<figure><img src="image_18.png" /><figcaption>The Palette widget of the iNavigator.</figcaption></figure>

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<figure><img src="image_19.png" /></figure>

A glycosaminoglycan Biomolecule report page

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<figure><img src="image_20.png" /></figure>

Building a three-dimensional model of a GAG sequence

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