Date on Master's Thesis/Doctoral Dissertation

8-2017

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Chemistry

Degree Program

Chemistry, PhD

Committee Chair

Maurer, Muriel

Committee Co-Chair (if applicable)

Merchant, Michael

Committee Member

Merchant, Michael

Committee Member

Mueller, Eugene

Committee Member

Wittebort, Ricard

Author's Keywords

fibrinogen; αC region; Factor XIII; intrinsically disordered proteins; mass spectrometry; NMR spectroscopy

Abstract

Fibrinogen is the most abundant protein involved in blood coagulation and has been associated with many pathological implications in cardiovascular disease. At the final stages of blood clot formation, the transglutaminase Factor XIIIa introduces γ-glutamyl-ε-lysinyl covalent bonds between reactive glutamines and lysines in fibrin, which results in a tighter clot network that is resistant to fibrinolysis. Factor XIIIa crosslinks specific reactive glutamines on fibrinogen, selecting more reactive glutamines in the αC region of fibrinogen than any other chain. Although crosslinking pairs in the αC region have been identified, little is known about the extent of crosslinking and the role played by each reactive glutamine. For the first time, we have ranked three reactive glutamines crosslinked under physiological conditions. A combination of MALDI-TOF mass spectrometry, LC-MS, and 2D 15N-1H HSQC NMR studies was used on Fibrinogen aC(233-425) to understand Factor XIIIa’s substrate specificity. Factor XIIIa’s specificity for each reactive glutamine in aC(233-425) was monitored by the enzyme's ability to cross-link a lysine mimic, glycine ethyl ester (GEE), to the reactive glutamines (Q237, Q328, Q366). We showed that Factor XIIIa crosslinks these three glutamines to GEE in the order Q237 >> Q366 ≈ Q328. Fibrinogen aC(233-425) variants were generated in which each reactive glutamine (Q) was replaced with an inactive asparagine (N). We demonstrated that Factor XIIIa still crosslinks each reactive glutamine independently and is more selective in the absence of the most reactive glutamine Q237. To examine the role of the putative Factor XIII binding site on αC(233-425), we performed experiments in which an important amino acid E396 was replaced with an alanine (Fbg αCE396A). Our results showed that both plasma Factor XIII A2B2 and recombinant Factor XIII A2 can crosslink all three reactive glutamines in Fbg αCE396A to a similar extent as with the WT. For structural characterization, aC(233-425) was expressed in 15N or 15N/13C-enriched media. 2D HSQC NMR experiments confirmed that the 15N-labeled αC(233-425) is mostly intrinsically disordered. Residues surrounding Q366 and Factor XIII's binding site αC(389-402) showed a higher propensity for order. 2D NMR experiments suggest the possibility of pre-structured motifs (PreSMOS) within Fibrinogen aC(233-425).

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