The protease allergen Der p 1 cleaves cell surface DC-SIGN and DC-SIGNR: experimental analysis of in silico substrate identification and implications in allergic responses.
BACKGROUND
The cysteine protease Der p 1 from the house dust mite Dermatophagoides pteronyssinus is one of the most potent allergens known. An attractive mechanism for a component of Der p 1 allergenicity lies in its ability to cleave key regulatory molecules from leucocyte surfaces, subverting cellular function and driving abnormal immunoglobulin E (IgE) responses.


OBJECTIVE
Although CD23, CD25 and CD40 have already been identified as major Der p 1 targets, other significant substrates may also exist.


METHODS
To investigate this, knowledge of the proteolytic properties of Der p 1 was used to perform in silico digestion of human dendritic cell surface proteins, using the prediction of protease specificity (PoPS) bioinformatics tool, in conjunction with cellular in vitro analysis and cleavage site determination.


RESULTS
Targets identified included DC-SIGN and DC-SIGNR, two C-type lectins implicated mostly in pathogen trafficking. Treatment of positively expressing cells with Der p 1 led to loss of detectable surface DC-SIGN and DC-SIGNR. Digestion of purified soluble recombinant DC-SIGN and DC-SIGNR, followed by N-terminal sequencing and MALDI mass spectrometry, indicated in each case one major cleavage site and several minor sites, the former correlating well with Der p 1 enzymology and the folded state of the substrate proteins. Loss of DC-SIGN from the cell surface led to reduced binding of intracellular adhesion molecule-3, an endogenous DC-SIGN ligand expressed on naïve T cells which is thought to be involved in T-helper type 1 cytokine signalling.


CONCLUSIONS
These data provide evidence of lectin involvement in the initiation of the allergic response and the value of using genome-wide in silico digestion tools.
Furmonaviciene
R
R
Institute of Infection, Immunity and Inflammation, School of Molecular Medical Sciences, University of Nottingham, Nottingham, UK.
Ghaemmaghami
A M
AM
Boyd
S E
SE
Jones
N S
NS
Bailey
K
K
Willis
A C
AC
Sewell
H F
HF
Mitchell
D A
DA
Shakib
F
F
D000485:Allergens / Q000276:immunology; D000818:Animals; D039741:Antigens, Dermatophagoides / Q000276:immunology*; D060829:Arthropod Proteins; D015815:Cell Adhesion Molecules / Q000276:immunology*; D003546:Cysteine Endopeptidases; D003713:Dendritic Cells / Q000276:immunology*; D006801:Humans; D037181:Lectins, C-Type / Q000276:immunology*; D051379:Mice; D008925:Mites / Q000276:immunology*; D011956:Receptors, Cell Surface / Q000276:immunology*; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin
D000485:Allergens; D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D015815:Cell Adhesion Molecules; D003546:Cysteine Endopeptidases; D037181:Lectins, C-Type; D011956:Receptors, Cell Surface; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin; C433029:CLEC4M protein, human; C451162:Dermatophagoides pteronyssinus antigen p 1