Figure 1.
Characteristics of the alternatively spliced transcript of VAP-1.
(A) Schematic presentation of the exon–intron organization of the human VAP-1 gene, AOC3. The boxes with roman numerals (I-IV) represent the exons. The translated regions are shown in color and the 5′- and 3′-untranslated regions in white. Exon III (violet) is spliced out in the shorter splice variant. Sv1: the full-length splice variant; Sv2: the alternatively spliced shorter splice variant. (B) Sequence alignment of the two VAP-1 isoforms. The deduced amino acid sequences of VAP-1 and the shorter isoform VAP-1Δ3. Highlighted are: light yellow, the hydrophobic N-terminal sequence; pink, the conserved signature motif of the active site, in which the first tyrosine is post-translationally modified to topaquinone; lilac, the (putative) catalytic site base; light green, the conserved Cu(II) binding histidine residues; light blue, the conserved cysteine residues involved in dimerization; orange the putative N-linked glycosylation sites, grey, RGD sequence. The amino acids unique to VAP-1Δ3 are in red.
Figure 2.
Expression of VAP-1Δ3 mRNA in adult and fetal human tissues.
(A) Commercial first strand cDNA panels were used to determine the mRNA expression of VAP-1Δ3 in respect to the expression of the full-length VAP-1 in adult human tissues by PCR. GAPDH expression was used as an endogenous control. (B) RT-PCR analysis of nine fetal human tissues was performed using VAP-1 and β-actin specific primers. The identity of the resulting amplicons was verified with Southern blotting using a VAP-1-specific probe. The two alternatively spliced mRNA species are marked with arrows. (C) qPCR analysis was performed with transcript specific primers and probes using the commercial first strand cDNA panels as templates. The expression levels of VAP-1 and VAP-1Δ3 are presented as percentages of β-actin mRNA expression in the same sample.
Figure 3.
Expression of VAP-1Δ3 in transiently transfected cell lines.
Flow cytometry of HEK293 cells transfected either with the full-length VAP-1- or with VAP-1Δ3 -cDNAs in pcDNA3.1 (A–B). The gray histograms: staining with the anti-VAP-1 polyclonal antibody; the black histograms: staining with a negative control antibody. The expression was also examined by fluorescence microscopy of acetone-permeabilized coverslip-plated HEK293 cells transfected with the corresponding constructs (C–D). In E–F, flow cytometry of HUVECs infected with pAdCMV-constructs of VAP-1- and VAP-1Δ3. The gray histograms: staining with the anti-VAP-1 polyclonal antibody; the black histograms: staining with a negative control antibody. The expression was also examined by fluorescence microscopy of acetone-permeabilized coverslip-plated HUVECs infected with the corresponding constructs (G–H). Scale bar 100 µm.
Figure 4.
Enzymatic activities of the two VAP-1 isoforms.
The SSAO-activity and substrate specificities of VAP-1 and VAP-1Δ3 were defined by a fluorometric assay from lysed transfectants. (A) HEK293 cells transfected with VAP-1. (B) HEK293 cells transfected with VAP-1Δ3 (C) HUVECs infected with VAP-1 (D) HUVECs infected with VAP-1Δ3. The final concentrations of all substrates were 1 mM. MA methylamine; BZ benzylamine; TYR p-tyramine; TRYPT tryptamine; PEA 2-phenylethylamine; HIS histamine. The results are expressed as nmol/mg/h+standard deviation (SD) (n = 3). (E) The structure of dimeric VAP-1 (PDB code 1US1; Airenne et al., 2002), viewed from the side of the carboxy-terminus along the two-fold axis. Both monomers are drawn in rainbow colors from blue amino-termini to red carboxy-termini. The amino acids missing from VAP-1Δ3 (aa 634-761) are illustrated as gray spheres of different shades. The picture was generated with PyMol [54].
Figure 5.
VAP-1 cell-surface expression and SSAO activity of transfectants expressing both VAP-1 and VAP-1 Δ3.
HUVECs were first infected with adenoviruses carrying the cDNA for the full-length VAP-1 and then with those carrying the VAP-1Δ3-cDNA. LacZ adenoviruses were used as co-transfection controls. VAP-1 expression was determined by FACS using the antibody JG 2.10. (A) The surface expression of VAP-1 in the transfected cells as mean fluorescence intensities (MFIs) and the averages of MFIs from three experiments. In parentheses, the percentage of VAP-1 surface expression in the co-transfected cells compared to the cells transfected only with VAP-1 ( = 100%). VAP-1Δ3 adenoinfection was performed with two different doses, 400 and 800 pfu (B) A histogram of a representative experiment. The number of cells is shown in the y-axis and the fluorescence in the x-axis. The green histogram shows VAP-1 surface expression in the cells co-infected with the control lacZ adenovirus, the blue histogram shows VAP-1 expression in the cells co-infected with VAP-1Δ3, and the red histogram shows the staining with a negative control antibody. (C) SSAO activity of stably transfected VAP-1-CHO cells co-transfected either with the EGFP-IRES2 empty vector (black) or with the same vector carrying VAP-1Δ3 (white). The enzymatic activity of lysed transfectants was determined in fluorometric assays. The substrates used were BZ benzylamine; MA methylamine (1 mM). Results are shown as nmol of H2O2/mg/h+SEM. The experiment was repeated four times with MA and five times with BZ.
Figure 6.
Heterodimerisation of VAP-1 and VAP-1 Δ3.
Lysates of HEK293 cells co-transfected with flag-tagged VAP-1 cDNA, myc-tagged VAP-1Δ3 cDNA or with the corresponding tagged empty vectors in different combinations were separated in SDS-PAGE (with or without prior immunoprecipitation) and blotted to nitrocellulose membranes. The functionality of the tagged constructs and the ability to detect the proteins with corresponding antibodies was first verified by using the flag-antibody (A) or the myc-antibody (B) in control gels without prior immunoprecipitations. Aliquots of the same lysates were then immunoprecipitated with the flag antibody prior to gel electrophoresis, and the immunoprecipitated product was detected using the myc-antibody (C). The sizes of the two VAP-1 isoforms and the molecular weight markers are indicated. Ip Immunoprecipitation, Ig Immunoglobulin.