In the extracellular environment, the enzyme transglutaminase 2 (TG2) is involved in cell-matrix interactions through association with the extracellular matrix protein, fibronectin (FN). The 45 kDa gelatin-binding domain of FN (45FN) is responsible for the binding to TG2. Previous studies have demonstrated that the FN-binding site of TG2 is located in the N-terminal domain of the enzyme although with conflicting results regarding the specific residues involved. Here we have mapped the FN interaction site of human TG2 by use of hydrogen/deuterium exchange coupled with mass spectrometry, and we confirm that the FN-binding site is located in the N-terminal domain of TG2. Furthermore, by combination of site-directed mutagenesis and surface plasmon resonance analysis we have identified the TG2 residues K30, R116 and H134 as crucial to maintain the high affinity interaction with FN. Mutation of all three residues simultaneously reduced binding to 45FN by more than 2000-fold. We also identified residues in the catalytic core domain of TG2 that contributed to FN binding, hence extending the binding interface between TG2 and FN. This study provides new insights into the high affinity interaction between TG2 and FN.

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Glutathione transferases from rat kidney cytosol were purified about 40-fold by chromatography on S-hexylglutathione linked to epoxy-activated Sepharose 6B. Further purification by fast protein liquid chromatography with chromatofocusing in the pH interval 10.6-7.6 resolved five major peaks of activity with 1-chloro-2,4-dinitrobenzene as the second substrate. Four of the peaks were identified with rat liver transferases 1-1, 1-2, 2-2 and 4-4 respectively. The criteria used for identification included physical properties, reactions with specific antibodies, substrate specificities and sensitivities to several inhibitors. The fourth major peak is a 'new' form of transferase, which has not been found in rat liver. This isoenzyme, glutathione transferase 7-7, has a lower apparent subunit Mr than any of the transferases isolated from rat liver cytosol, and does not react with antibodies raised against the liver enzymes. Glutathione transferases 3-3 and 3-4, which are abundant in liver, were only present in very small amounts. In a separate chromatofocusing separation in a lower pH interval, an additional peak was eluted at pH 6.3. This isoenzyme is characterized by its high activity with ethacrynic acid.

1Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become an important method 2 to study the structural dynamics of proteins. However, glycoproteins represent a challenge to the traditional 3 HDX-MS workflow for determining the deuterium uptake of the protein segments that contain the glycan. We 4 have recently demonstrated the utility of the glycosidase PNGase A to enable HDX-MS analysis of N-glycosylated 5 protein regions. Here we have investigated the use of the acidic glycosidase PNGase H + , which has a pH optimum 6 at 2.6, to efficiently deglycosylate N-linked glycosylated peptides during HDX-MS analysis of glycoproteins. Our 7 results show that PNGase H + retains high deglycosylation activity at HDX quench conditions. When used in an 8 HDX-MS workflow, PNGase H + allowed the extraction of HDX data from all five glycosylated regions of the serpin 9 α1-antichymotrypsin. We demonstrate that PNGase A and PNGase H + are capable of similar deglycosylation 10 performance during HDX-MS analysis of α1-antichymotrypsin and the IgG1 antibody Trastuzumab (TZ). However, 11PNGase H + provides broader specificity and greater tolerance to the disulfide-bond reducing agent TCEP, while 12

Antithrombin deficiency is associated with increased risk of venous thrombosis. In certain families, this condition is caused by pathogenic polymerization of mutated antithrombin in the blood. To facilitate future development of pharmaceuticals against antithrombin polymerization, an improved understanding of the polymerogenic intermediates is crucial. However, X-ray crystallography of these intermediates is severely hampered by the difficulty in obtaining well-diffracting crystals of transient and heterogeneous noncovalent protein assemblies. Furthermore, their large size prohibits structural analysis by NMR spectroscopy. Here, we show how hydrogen/deuterium-exchange mass spectrometry (HDX-MS) provides detailed insight into the structural dynamics of each subunit in a polymerization-competent antithrombin dimer. Upon deuteration, this dimer surprisingly yields bimodal isotope distributions for the majority of peptides, demonstrating an asymmetric configuration of the two subunits. The data reveal that one subunit is very dynamic, potentially intrinsically disordered, whereas the other is considerably less dynamic. The local subunit-specific deuterium uptake of this polymerization-competent dimer strongly supports a β4A-β5A β-hairpin runaway domain swap mechanism for antithrombin polymerization. HDX-MS thus holds exceptional promise as an enabling analytical technique in the efforts toward future pharmacological intervention with protein polymerization and associated diseases.