Mapping the topographic epitope landscape on the urokinase plasminogen activator receptor (uPAR) by surface plasmon resonance and X-ray crystallography.
The urokinase-type plasminogen activator receptor (uPAR or CD87) is a glycolipid-anchored membrane protein often expressed in the microenvironment of invasive solid cancers and high levels are generally associated with poor patient prognosis (Kriegbaum et al., 2011 [1]). uPAR is organized as a dynamic modular protein structure composed of three homologous Ly6/uPAR domains (LU).This internally flexible protein structure of uPAR enables an allosteric regulation of the interactions with its two principal ligands: the serine protease urokinase-type plasminogen activator (uPA) and the provisional matrix protein vitronectin (Vn) (Mertens et al., 2012; Gårdsvoll et al., 2011; Madsen et al., 2007 [2-4]). The data presented here relates to the non-covalent trapping of one of these biologically relevant uPAR-conformations by a novel class of monoclonal antibodies (Zhao et al., 2015 [5]) and to the general mapping of the topographic epitope landscape on uPAR. The methods required to achieve these data include: (1) recombinant expression and purification of a uPAR-hybrid protein trapped in the desired conformation [patent; WO 2013/020898 A12013]; (2) developing monoclonal antibodies with unique specificities using this protein as antigen; (3) mapping the functional epitope on uPAR for these mAbs by surface plasmon resonance with a complete library of purified single-site uPAR mutants (Zhao et al., 2015; Gårdsvoll et al., 2006 [5,6]); and finally (4) solving the three-dimensional structures for one of these mAbs by X-ray crystallography alone and in complex with uPAR [deposited in the PDB database as 4QTH and 4QTI, respectively].
State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China ; Danish-Chinese Centre for Proteases and Cancer.
Baoyu
B
Zhao
FIRC Institute of Molecular Oncology, Via Adamello 16, 20139 Milan, Italy.
Sonu
S
Gandhi
State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China ; Danish-Chinese Centre for Proteases and Cancer.
Cai
C
Yuan
State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China ; Danish-Chinese Centre for Proteases and Cancer.
Zhipu
Z
Luo
State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China ; Biotech Research and Innovation Centre (BRIC), Copenhagen Biocenter, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark.
Rui
R
Li
Finsen Laboratory, Rigshospitalet, Copenhagen Biocenter, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark ; Biotech Research and Innovation Centre (BRIC), Copenhagen Biocenter, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark ; Danish-Chinese Centre for Proteases and Cancer.
Henrik
H
Gårdsvoll
FIRC Institute of Molecular Oncology, Via Adamello 16, 20139 Milan, Italy.
Valentina
V
de Lorenzi
FIRC Institute of Molecular Oncology, Via Adamello 16, 20139 Milan, Italy.
Nicolai
N
Sidenius
State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China ; Danish-Chinese Centre for Proteases and Cancer.
Mingdong
M
Huang
Finsen Laboratory, Rigshospitalet, Copenhagen Biocenter, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark ; Biotech Research and Innovation Centre (BRIC), Copenhagen Biocenter, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark ; Danish-Chinese Centre for Proteases and Cancer.
Michael
M
Ploug
Allostery; CD87; Cancer invasion; Epitope mapped antibodies; Hot spots; SPR; Vitronectin; uPAR