Post-translational modification of type IV collagen with 3-hydroxyproline affects its interactions with glycoprotein VI and nidogens 1 and 2

• Published in: The Journal of biological chemistry Vol. 293; no. 16; pp. 5987 - 5999
• Main Authors: Montgomery, Nathan T., Zientek, Keith D., Pokidysheva, Elena N., Bächinger, Hans Peter
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 20.04.2018; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; basement membrane; Calcium-Binding Proteins; Cell Adhesion Molecules; Cell Line; collagen; collagen IV; Collagen Type IV - metabolism; Glycobiology and Extracellular Matrices; Hydroxyproline - metabolism; mass spectrometry (MS); Membrane Glycoproteins - metabolism; Mice; Platelet Membrane Glycoproteins - metabolism; post-translational modification (PTM); protein chemistry; Protein Interaction Maps; protein interactions; Protein Processing, Post-Translational; tandem mass spectrometry; post-translational modification (PTM); protein interactions; basement membrane; tandem mass spectrometry; collagen; protein chemistry; collagen IV; mass spectrometry (MS)
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA117.000406

Type IV collagen is a major component of the basement membrane and interacts with numerous other basement membrane proteins. Many of these interactions are poorly characterized. Type IV collagen is abundantly post-translationally modified with 3-hydroxyproline (3-Hyp), but 3-Hyp’s biochemical role in type IV collagen’s interactions with other proteins is not well established. In this work, we present binding data consistent with a major role of 3-Hyp in interactions of collagen IV with glycoprotein VI and nidogens 1 and 2. The increased binding interaction between type IV collagen without 3-Hyp and glycoprotein VI has been the subject of some controversy, which we sought to explore, whereas the lack of binding of nidogens to type IV collagen without 3-Hyp is novel. Using tandem MS, we show that the putative glycoprotein VI-binding site is 3-Hyp–modified in WT PFHR-9 type IV collagen, but not in PFHR-9 cells in which prolyl-3-hydroxylase 2 (P3H2) has been knocked out (KO). Moreover, we observed altered 3-Hyp occupancy across many other sites. Using amino acid analysis of type IV collagen from the WT and P3H2 KO cell lines, we confirm that P3H2 is the major, but not the only 3-Hyp–modifying enzyme of type IV collagen. These findings underscore the importance of post-translational modifications of type IV collagen for interactions with other proteins.