A contaminant trypsin-like activity from the timothy grass pollen is responsible for the conflicting enzymatic behavior of the major allergen Phl p 1

作者：Baeyens Volant Danielle; M'Rabet Nasiha; El Mahyaoui Rachida; Wattiez Ruddy; Azarkan Mohamed^*

来源：Biochimica et Biophysica Acta-Proteins and Proteomics, 2013, 1834(1): 272-283.

DOI：10.1016/j.bbapap.2012.06.012

• Phleum pratense
• Group-1 allergen
• beta-expansin
• Serine protease
• Phl p 1

We intend to solve whether or not Phi p 1 can be regarded as a protease. A group reported that Phi p I has papain-like properties and later on, that this allergen resembles cathepsin B, while another one demonstrated that Phi p 1 lacks proteinase activity and suggested that the measured activity may rise either from a recombinant Phl p 1 contaminant or as a result of an incompletely purified natural allergen. A third group reported Phl p 1 to act by a non-proteolytic activity mechanism. We report the purification of the natural Phl p I by means of hydrophobic interaction, gel filtration and STI-Sepharose affinity chromatographies. The Phi p 1 purity was assessed by silver-stained SDS-PAGE and by 'in-gel' and 'gel-free' approaches associated to mass spectrometry analyses. The proteolytic activity was measured using Boc-Gln-Ala-Arg-AMC and Z-Phe-Arg-AMC as substrates. While amidolytic activity could be measured with Phl p 1 after rechromatography on gel filtration, it however completely disappeared after chromatography on STI-Sepharose. The contaminant activity co-eluting with Phi p 1 was not affected by cysteine proteases inhibitors and other thiol-blocking agents, by metalloproteases inhibitors and by aspartic proteases inhibitors. However, it was completely inhibited by low molecular weight and proteinaceous serine proteases inhibitors. TLCK, but not TPCK, inhibited the contaminant activity, showing a trypsin-like behavior. The pH and temperature optimum were 8.0 and 37 degrees C, respectively. These data indicated that Phi p 1 is not a protease. The contaminant trypsin-like activity should be considered when Phl p 1 allergenicity is emphasized.

• 出版日期2013-1