On the localization of the cleavage site in human alpha-2-antiplasmin, involved in the generation of the non-plasminogen binding form

Abstract

Background: Alpha-2-antiplasmin (α2AP) is the main natural inhibitor of plasmin. The C-terminus of α2AP is crucial for the initial interaction with plasmin(ogen) and the rapid inhibitory mechanism. Approximately 35% of circulating α2AP has lost its C-terminus (non-plasminogen binding α2AP/NPB-α2AP) and thereby its rapid inhibitory capacity. The C-terminal cleavage site of α2AP is still unknown. A commercially available monoclonal antibody against α2AP (TC 3AP) detects intact but not NPB-α2AP, suggesting that the cleavage site is located N-terminally from the epitope of TC 3AP.

Objectives: To determine the epitope of TC 3AP and then to localize the C-terminal cleavage site of α2AP.

Methods: For epitope mapping of TC 3AP, commercially available plasma purified α2AP was enzymatically digested with Asp-N, Glu-C, or Lys-N. The resulting peptides were immunoprecipitated using TC 3AP-loaded Dynabeads® Protein G. Bound peptides were eluted and analyzed by liquid chromatography-tandem mass spectometry (LC-MS/MS). To localize the C-terminal cleavage site precisely, α2AP (intact and NPB) was purified from plasma and analyzed by LC-MS/MS after enzymatic digestion with Arg-C.

Results: We localized the epitope of TC 3AP between amino acid residues Asp428 and Gly439. LC-MS/MS data from plasma purified α2AP showed that NPB-α2AP results from cleavage at Gln421-Asp422 as preferred site, but also after Leu417, Glu419, Gln420, or Asp422.

Conclusions: The C-terminal cleavage site of human α2AP is located N-terminally from the TC 3AP epitope. Because C-terminal cleavage of α2AP can occur after multiple residues, different proteases may be responsible for the generation of NPB-α2AP.

Keywords: alpha-2-antiplasmin; epitope mapping; mass spectrometry; proteolysis; western blot.

Figure 1

In‐house purified α2AP visualized by SDS‐PAGE and Western blot. (A) SDS‐gel of purified α2AP. (B) Immunoblot obtained with anti‐Asn‐α2AP. (C) Immunoblot obtained with TC 3AP. (D) Merged anti‐Asn‐α2AP and TC 3AP blots. The relative migration distances of molecular weight marker proteins are indicated on the left. PB‐α2AP is indicated by arrowhead. NPB‐α2AP is indicated by an asterisk, as well as by a red box in (A)

Figure 2

MS/MS fragmentation spectra of the (A) nondeamidated and (B) deamidated Asn408‐Gln421 peptide in an overnight Arg‐C digest of NPB‐α2AP as determined by Mascot and MaxQuant analysis, respectively. Best spectrum data are shown. The overlapping labels of the peaks between m/z 730 and 750 in (A) should be read as the following: b(13)++ m/z 731.3759; y0(13)++,y*(13)++, z + 1(13)**, m/z 738.3837, 738.8757, 739.3796; y(13)++ m/z 747.3890

Figure 3

Three‐dimensional structure of human α2AP as predicted by I‐TASSER.27 Red, β‐sheet A; green, β‐sheet B; yellow, β‐sheet C; magenta, reactive center loop; cyan, C‐terminal cleavage area Leu417‐Asp422 (LKEQQD); blue, C‐terminus; orange, epitope of TC 3AP. Left panel: front view. Right panel: side view, 90°C clockwise rotation

Figure 4

Schematic representation of the α2AP C‐terminus from Asn408 to Lys464. Arrows indicate the in vivo C‐terminal cleavage sites (preferred site between Gln421 and Asp422), the bold underlined sequence represents the area that includes the TC 3AP epitope, and the brace indicates the area that includes the in vitro C‐terminal cleavage site as described by Sasaki et al.17