title: A contaminant trypsin-like activity from the timothy grass pollen is responsible for the conflicting enzymatic behavior of the major allergen Phl p 1.
abstract: We intend to solve whether or not Phl p 1 can be regarded as a protease. A group reported that Phl p 1 has papain-like properties and later on, that this allergen resembles cathepsin B, while another one demonstrated that Phl 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 1 by means of hydrophobic interaction, gel filtration and STI-Sepharose affinity chromatographies. The Phl 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 Phl 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°C, respectively. These data indicated that Phl p 1 is not a protease. The contaminant trypsin-like activity should be considered when Phl p 1 allergenicity is emphasized.
authors: University of Brussels, Faculty of Medicine, Protein Chemistry Unit, Campus Erasme (CP 609), 808, route de Lennik, B-1070 Brussels, Belgium.
authors: Danielle
authors: D
authors: Baeyens-Volant
authors: Nasiha
authors: N
authors: M'Rabet
authors: Rachida
authors: R
authors: El Mahyaoui
authors: Ruddy
authors: R
authors: Wattiez
authors: Mohamed
authors: M
authors: Azarkan
mesh_terms: D000485:Allergens / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D031784:Phleum / Q000737:chemistry* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000485:Allergens; D010940:Plant Proteins; C090396:PHLPI protein, Phleum pratense; D014357:Trypsin