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. 2006 Dec 8;281(49):37877-87.
doi: 10.1074/jbc.M609398200. Epub 2006 Oct 17.

Detection and Purification of Tyrosine-Sulfated Proteins Using a Novel Anti-Sulfotyrosine Monoclonal Antibody

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Free PMC article

Detection and Purification of Tyrosine-Sulfated Proteins Using a Novel Anti-Sulfotyrosine Monoclonal Antibody

Adam J Hoffhines et al. J Biol Chem. .
Free PMC article

Abstract

Protein tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST1 and TPST2) that catalyze the transfer of sulfate to tyrosine residues in secreted and transmembrane proteins. Tyrosine sulfation plays a role in protein-protein interactions in several well defined systems. Although dozens of tyrosine-sulfated proteins are known, many more are likely to exist and await description. Advancing our understanding of the importance of tyrosine sulfation in biological systems requires the development of new tools for the detection and study of tyrosine-sulfated proteins. We have developed a novel anti-sulfotyrosine monoclonal antibody (called PSG2) that binds with high affinity and exquisite specificity to sulfotyrosine residues in peptides and proteins independently of sequence context. We show that it can detect tyrosine-sulfated proteins in complex biological samples and can be used as a probe to assess the role of tyrosine sulfation in protein function. We also demonstrate the utility of PSG2 in the purification of tyrosine-sulfated proteins from crude tissue samples. Finally, Western blot analysis using PSG2 showed that certain sperm/epididymal proteins are undersulfated in Tpst2(-/-) mice. This indicates that TPST1 and TPST2 have distinct macromolecular substrate specificities and provides clues as to the molecular mechanism of the infertility of Tpst2(-/-) males. PSG2 should be widely applicable for identification of tyrosine-sulfated proteins in other systems and organisms.

Figures

FIGURE 1
FIGURE 1
PSG2 Epitope Mapping. Peptides were synthesized on PEG-modified cellulose membranes and PSG2 binding was assessed by Western blotting as described in Experimental Procedures. A. Single amino acid substitution analysis was performed on two pentapeptides modeled on the N-terminus of human PSGL-1. The first and last columns are the respective sulfated peptides. In the other columns, each position was substituted with the amino acid indicated in single letter code. B. Sixty-seven pentapeptide sequences selected using a random sequence generator were synthesized with either sulfotyrosine (columns labeled a) or tyrosine (columns labeled b) in the third position. The sequences of the peptides on the array are shown in Supplemental Table 1.
FIGURE 2
FIGURE 2
PSG2 Western Blotting of Purified Tyrosine-Sulfated Proteins. Bovine factor X1 and X2 (panel A) and human C4 (panel B) were run on 4-15% SDS-polyacrylamide gels and stained with Coomassie blue (CB) or transferred to PVDF and subjected to Western blotting with PSG2 as described in Experimental Procedures. In lanes stained with Coomassie blue (CB) 1 μg of the proteins were loaded. In lanes subjected to Western blotting 10 ng of factors X1 and X2 (0.18 pmol) or C4 (0.05 pmol) were loaded. HC = heavy chain.
FIGURE 3
FIGURE 3
PSG2 Western Blotting of Cell and Tissue Extracts. A. Detergent extracts of wild type and Tpst double knockout liver (10 μg total protein) were run on 4-15% SDS gels and stained with Coomassie blue (CB) or transferred to PVDF and subjected to Western blotting with PSG2 as described in Experimental Procedures. B. Rat NRK cells were treated with buffer (lanes 1) or 100 μM pervanadate (lanes 2). The cells were solubilized in detergent, run on 4-15% SDS gels, transferred to PVDF, and subjected to Western blotting with the anti-phosphotyrosine mAb PY20 or PSG2 as described in Experimental Procedures.
FIGURE 4
FIGURE 4
PSG2 Blocks Neutrophil Adhesion to P-selectin Under Flow. P-selectin was coated onto plates and neutrophils were perfused over the surface at 1 dyn/cm2. Adhesion was allowed to equilibrate and then non-adherent cells were flushed from the system. Rolling neutrophils were counted every 20 s. In one experiment (red line) control human IgG4-λ (50 μg/ml) and then PSG2 (50 μg/ml) was sequentially added to the perfusate at the indicated time points. In the other experiment (blue line) PSG2 was added to the perfusate first.
FIGURE 5
FIGURE 5
Affinity Purification of Proteins From Mouse Epididymis. Proteins from the soluble fraction of wild type mouse epididymis were applied to a PSG2 affinity column and the column was eluted with 4 mM LD(sY)DF peptide. Early (lanes 1) and late (lanes 2) fractions from the peptide elution were run on 4-15% SDS-polyacrylamide gels which were then either silver stained (left panel) or transferred to PVDF membrane and probed with PSG2 (right panel). The bands subjected to in-gel tryptic digestion and LC-MS/MS sequencing are labeled (A-D).
FIGURE 6
FIGURE 6
LC-MS/MS Sequencing. A. Total ion chromatogram (TIC) of the tryptic digest from the protein gel band B shown in Figure 5. B. FTICR mass spectrum of peptides eluted in the first 32 minutes of the LC gradient. The insert shows the MASCOT histogram of the score distribution for the proteins identified in the gel band B. C. MS2 scan of the m/z 1026.98 ion. The insert shows the precursor mass scan at 20.97 min using the FTICR (single scan from 300 to 1400 m/z with 100,000 resolution at 106 target ions). For illustration purposes, only the 1026 to 1029 m/z mass region of the [M+2H]2+ ion is shown. D. Peptide sequence (154-170) from the fibrinogen β chain identified by MASCOT and manually validated from the MS2 spectrum of the m/z 1026.98 ion.
FIGURE 7
FIGURE 7
PSG2 Western Blots of Mouse Fibrinogen. Purified mouse fibrinogen was run on 4-15% SDS gels, transferred to PVDF, and subjected to Western blotting with PSG2 as described in Experimental Procedures. In lanes stained with Coomassie blue (CB) 2 μg of protein were loaded. In lanes subjected to Western blotting with PSG2, 10 ng was loaded.
FIGURE 8
FIGURE 8
PSG2 Western Blotting of Epididymal/Sperm Proteins. Detergent extracts of whole epididymis from wild type, Tpst1−/−, and Tpst2−/− mice (15 μg total protein) were run on 4-15% SDS-polyacrylamide gels and stained with Coomassie blue (CB) or transferred to PVDF and subjected to Western blotting with PSG2 as described in Experimental Procedures.

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