Mass Spectrometry-Based Disulfide Mapping of Lysyl Oxidase-like 2

Abstract

Lysyl oxidase-like 2 (LOXL2) catalyzes the oxidative deamination of peptidyl lysines and hydroxylysines to promote extracellular matrix remodeling. Aberrant activity of LOXL2 has been associated with organ fibrosis and tumor metastasis. The lysine tyrosylquinone (LTQ) cofactor is derived from Lys653 and Tyr689 in the amine oxidase domain via post-translational modification. Based on the similarity in hydrodynamic radius and radius of gyration, we recently proposed that the overall structures of the mature LOXL2 (containing LTQ) and the precursor LOXL2 (no LTQ) are very similar. In this study, we conducted a mass spectrometry-based disulfide mapping analysis of recombinant LOXL2 in three forms: a full-length LOXL2 (fl-LOXL2) containing a nearly stoichiometric amount of LTQ, Δ1-2SRCR-LOXL2 (SRCR1 and SRCR2 are truncated) in the precursor form, and Δ1-3SRCR-LOXL2 (SRCR1, SRCR2, SRCR3 are truncated) in a mixture of the precursor and the mature forms. We detected a set of five disulfide bonds that is conserved in both the precursor and the mature recombinant LOXL2s. In addition, we detected a set of four alternative disulfide bonds in low abundance that is not associated with the mature LOXL2. These results suggest that the major set of five disulfide bonds is retained post-LTQ formation.

Keywords: disulfide bonds; lysine tyrosylquinone; lysyl oxidase-like 2; mass spectrometry.

Figure 1

A schematic representation of LOX and forms of LOXL2 used in this study.

Figure 2

Structures of the Zn(II)-bound precursor form of LOXL2 (PDB: 5ZE3) and the 3D homology modeled Cu(II)-bound mature form of LOX (PDB: ao9b00317_si_002.pdb in [17]). (a) The amine oxidase domain of LOXL2. The precursor residues (Lys653 and Tyr689) of the LTQ cofactor are in magenta. A Zn(II) (in navy sphere) occupies the predicted Cu(II) binding site, His626-X-His628-X-His630 motif (in green). There are five disulfide bonds (in yellow) in the amine oxidase domains of LOXL2 and LOX, but four out of five cysteine pairs are different (Table 1). (b) The amine oxidase domain of LOX where the LTQ structure (Lys320 + resi355) was modeled in. Cu(II) (in cyan sphere) is near the His292-X-His294-X-His296 motif (in green). (c) The ϵ-amino group of Lys653 is located at 16.6. Å distance from C2 of Tyr689 in the precursor LOXL2 structure. Zn(II) is in tetrahedral coordination geometry. (d) The o-quinone moiety of the LTQ cofactor is ligated to Cu(II) (2.1 Å, solid lines in cyan) but three His residues are >3 Å away from Cu(II) in the 3D-modeled LOX structure (dashed lines in cyan).

Figure 3

The sequence alignment of the C-terminal catalytic domain of the LOX-family of proteins. The residue numbers are those of LOXL2. Conserved residues are in bold. The His-X-His-X-His motif for Cu(II)-binding is in green. The precursor residues (Lys and Tyr) of the LTQ cofactor are in pink. Conserved Cys residues are in orange. The multiple sequence alignment was conducted by COBALT [19].

Figure 4

UV-vis spectroscopic titration of the LTQ cofactor by 2HP. (a) UV-vis spectral change during the 2HP titration of fl-LOXL2. (b) A plot of absorbance change at 531 nm (baseline subtracted) versus the molar ratio of 2HP over fl-LOXL2. (c) UV-vis spectral change during the 2HP titration of Δ1-3SRCR-LOXL2. (d) A plot of absorbance change at 531 nm (baseline subtracted) versus the molar ratio of 2HP over Δ1-3SRCR-LOXL2.

Figure 5

The oxidation of cadaverine by fl-LOXL2 (●) and Δ1-3SRCR-LOXL2 (○) follows the Michaelis–Menten kinetics at pH 8.0. The equation used for non-linear curve fitting to obtain the kinetic parameters is: V = {(k_cat [S])/((K_M + [S]) )} × [E_T], where E_T: total enzyme concentration, [S]: substrate concentration, k_cat: turnover K_M: Michaelis–Menten constant. The assay was performed in triplicate and data were plotted as the mean of three data points with standard deviation.

Figure 6

MS/MS data confirming the presence of peptide no. 12. (a) ETD data for the peptide containing LTQ cofactor (peptide no. 12b). (b) ETD data for the peptide without the LTQ cofactor (peptide no. 12a).

Figure 7

Quantitative comparison peptide no. 18 (sequence is shown in Table 5) in fl-LOXL2 (Table S6) and Δ1-2SRCR-LOXL2 (Table S5). (a) The XIC of peptide no. 18 (m/z 893) and a co-eluting peptide (m/z 717), used as an internal standard, for fl-LOXL2. (b) The mass spectrum generated from the LC–MS in (a) when both peaks are eluting. (c) The XIC of peptide no. 18 and its coeluting internal standard for the Δ1-2SRCR-LOXL2. (d) The mass spectrum generated from the LC–MS data in (c) when both peptides are present. By comparing the chromatograms in (a,c), one can clearly observe that the abundance of peptide no. 18 does not appreciably change with respect to the internal standard peptide, m/z 717, when the construct changes from fl-LOXL2 to Δ1-2SRCR-LOXL2.