title: Determination of Histidine pKa Values in the Propeptides of Furin and Proprotein Convertase 1/3 Using Histidine Hydrogen-Deuterium Exchange Mass Spectrometry.
abstract: Propeptides of proprotein convertases regulate activation of their protease domains by sensing the organellar pH within the secretory pathway. Earlier experimental work highlighted the importance of a conserved histidine residue within the propeptide of a widely studied member, furin. A subsequent evolutionary analysis found an increase in histidine content within propeptides of secreted eukaryotic proteases compared with their prokaryotic orthologs. However, furin activates in the trans-golgi network at a pH of 6.5 while a paralog, proprotein convertase 1/3, activates in secretory vesicles at a pH of 5.5. It is unclear how a conserved histidine can mediate activation at two different pH values. In this manuscript, we measured the pKa values of histidines within the propeptides of furin and proprotein convertase 1/3 using a histidine hydrogen-deuterium exchange mass spectrometry approach. The high density of histidine residues combined with an abundance of basic residues provided challenges for generation of peptide ions with unique histidine residues, which were overcome by employing ETD fragmentation. During this analysis, we found slow hydrogen-deuterium exchange in residues other than histidine at basic pH. Finally, we demonstrate that the pKa of the conserved histidine in proprotein convertase 1/3 is acid-shifted compared with furin and is consistent with its lower pH of activation.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, United States.
authors.forename: Johannes
authors.initials: J
authors.lastname: Elferich
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, United States.
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Williamson
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, United States.
authors.forename: Larry L
authors.initials: LL
authors.lastname: David
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, United States.
authors.forename: Ujwal
authors.initials: U
authors.lastname: Shinde
mesh_terms: D000595:Amino Acid Sequence; D003903:Deuterium / Q000737:chemistry; D045683:Furin / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D045664:Proprotein Convertase 1 / Q000737:chemistry*; D043484:Proprotein Convertases / Q000737:chemistry*
chemical_list: D010455:Peptides; D006639:Histidine; D006859:Hydrogen; D003903:Deuterium; D043484:Proprotein Convertases; D045683:Furin; D045664:Proprotein Convertase 1