title: Basic Residues of β-Sheet A Contribute to Heparin Binding and Activation of Vaspin (Serpin A12).
abstract: Many members of the serine protease inhibitor (serpin) family are activated by glycosaminoglycans (GAGs). Visceral adipose tissue-derived serpin (vaspin), serpin A12 of the serpin family, and its target protease kallikrein 7 (KLK7) are heparin-binding proteins, and inhibition of KLK7 by vaspin is accelerated by heparin. However, the nature of GAG binding to vaspin is not known. Here, we measured vaspin binding of various glycosaminoglycans and low molecular weight heparins by microscale thermophoresis and analyzed acceleration of protease inhibition by these molecules. In addition, basic residues contributing to heparin binding and heparin activation were identified by a selective labeling approach. Together, these data show that vaspin binds heparin with high affinity (KD= 21 ± 2 nm) and that binding takes place at a basic patch on top of β-sheet A and is different from other heparin-binding serpins. Mutation of basic residues decreased heparin binding and activation of vaspin. Similarly, reactive center loop insertion into sheet A decreased heparin binding because it disturbs the basic cluster. Finally, using vaspin-overexpressing keratinocyte cells, we show that a significant part of secreted vaspin is bound in the extracellular matrix on the cell surface. Together, basic residues of central β-sheet A contribute to heparin binding and activation of vaspin. Thus, binding to GAGs in the extracellular matrix can direct and regulate vaspin interaction with target proteases or other proteins and may play an important role in the various beneficial functions of vaspin in different tissues.
authors.affiliation: From the Institute of Biochemistry, Faculty of Biosciences, Pharmacy, and Psychology.
authors.forename: David
authors.initials: D
authors.lastname: Ulbricht
authors.affiliation: From the Institute of Biochemistry, Faculty of Biosciences, Pharmacy, and Psychology.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Oertwig
authors.affiliation: From the Institute of Biochemistry, Faculty of Biosciences, Pharmacy, and Psychology.
authors.forename: Kristin
authors.initials: K
authors.lastname: Arnsburg
authors.affiliation: the Department of Dermatology, Venerology, and Allergology, and.
authors.forename: Anja
authors.initials: A
authors.lastname: Saalbach
authors.affiliation: the Center for Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, University of Leipzig, 04103 Leipzig, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Pippel
authors.affiliation: the Center for Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, University of Leipzig, 04103 Leipzig, Germany.
authors.forename: Norbert
authors.initials: N
authors.lastname: Sträter
authors.affiliation: From the Institute of Biochemistry, Faculty of Biosciences, Pharmacy, and Psychology, jheiker@uni-leipzig.de.
authors.forename: John T
authors.initials: JT
authors.lastname: Heiker
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D005109:Extracellular Matrix / Q000737:chemistry / Q000378:metabolism; D006493:Heparin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D015843:Serpins / Q000737:chemistry / Q000378:metabolism
chemical_list: C507339:SERPINA12 protein, human; D015843:Serpins; D006493:Heparin
keywords: adipokine; glycosaminoglycan; heparin-binding protein; kallikrein 7; protease inhibitor; serine protease; serpin; structural biology; vaspin