Structural Mechanisms of Inactivation in Scabies Mite Serine Protease Paralogues

https://doi.org/10.1016/j.jmb.2009.04.082Get rights and content

Abstract

The scabies mite (Sarcoptes scabiei) is a parasite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to those in the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed scabies mite inactivated protease paralogues (SMIPPs). The precise function of SMIPPs is unclear; however, it has been suggested that these proteins might function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 Å and 2.0 Å resolution, respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site-directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical “canonical” fashion, and instead have evolved alternative functions in the lifecycle of the scabies mite.

Introduction

Scabies is a parasitic infection of the skin caused by the burrowing of the ectoparasitic itch mite Sarcoptes scabiei.1, 2, 3 The disease afflicts about 300 million people worldwide and is particularly problematic within socially disadvantaged populations. Infections are endemic in Aboriginal communities in Northern Australia that are characterized by inadequate medical facilities and overcrowding.4, 5 Scabies is prevalent also in immuno-deficient patients and in residents of nursing homes, despite the availability of chemotherapy control measures.6 Infestation occurs when the adult female mite burrows into the skin. Pruritic scabies lesions are often accompanied by opportunistic streptococcal infections with significant sequelae (cellulitis, septicemia and glomerulonephritis) and the increased community streptococcal burden contributes to extreme levels of rheumatic fever and rheumatic heart disease.4, 5 The wide prevalence and decreasing efficacy of treatment for scabies indicate that development of novel control strategies, in the form of vaccination or immunotherapy, are warranted; this is feasible as clinical immunity to scabies develops in humans.7, 8 A comprehensive review of these aspects was published recently.9

The paucity of molecular data on scabies is due to the difficulty of obtaining mites in sufficient numbers, because the parasite burden is generally very low and no in vitro culture system for propagating mites is available. In order to address this problem, using scabies mites from skin shed into the bedding of severe crusted scabies patients, we recently created a sequence database and identified gene homologues to major allergens of house dust mites.9, 10, 11 In particular, 33 genes homologous to the serine protease group 3 major allergen were identified. Remarkably, with one exception, all proteins contain mutations in the catalytic triad. This suggests strongly that members of this family cannot act as proteases by a known mechanism, and they have accordingly been named scabies mite inactivated protease paralogues (SMIPPs).12 Here, we present the result of a structural, biochemical and phylogenetic investigation of the SMIPPs. The crystal structures of two members reveal considerable structural rearrangements within and around the active site. This provides an explanation for the absence of protease function, and suggests that, in contrast to active serine proteases, these molecules are unable to bind substrates in a canonical fashion.13

Section snippets

SMIPPs adopt the chymotrypsin-like serine protease fold

Both SMIPPs crystallised as a dimer in the asymmetric unit of the crystal, with distinct relative orientations. There is no experimental evidence, however, to support the presence or physiological relevance of the dimeric form, and therefore we conclude that the dimers are crystalline artefacts. Both structures adopt the chymotrypsin-like serine protease fold, with residues 12–229 and 1–235 visible in the electron density for SMIPP-S-I1 and SMIPP-S-D1, respectively. There was no significant

Discussion

Although the proteolytically active member of the family, SARS3-A1, exhibits the hallmarks of an active serine protease, the unique sequence and structural features of the SMIPP family discussed above suggest strongly that this family functions in a way that is distinctly different from that of a catalytically active serine protease. Given the clear sequence homology with house dust mite serine protease allergens, and the knowledge that scabies infestation elicits a delayed-type

Expression of SMIPP protein in Pichia pastoris and purification

SMIPP-S-I1 and SMIPP-S-D1 were produced in Pichia pastoris. To direct secretion of the expressed protein into the medium, all constructs placed the predicted N-terminus flush with the Kex2 signal peptide cleavage site in the transfer vector pPICZalphaA (Invitrogen Australia Pty Limited, VIC, Australia). A stop codon at the 3′ end of the SMIPP sequence was introduced to ensure that the recombinant protein did not include any of the C-terminal tags of the vector. The plasmid was propagated in

Acknowledgements

A.M.B is a National Health and Medical Research Council of Australia Senior Research Fellow. J.C.W. is an Australian Research Council Federation Fellow and honorary NHMRC Principal Research Fellow. D. J. K. is an NHMRC Senior Principal Research Fellow. J.A.I is an NHMRC CJ Martin Postdoctoral Fellow. This work was supported by the NHMRC, the Australian Research Council and the State Government of Victoria (Australia). We thank the staff at IMCA-CAT (The Advanced Photon Source, Chicago) for

References (36)

  • CurrieB.J. et al.

    Skin infections and infestations in Aboriginal communities in northern Australia

    Australas J. Dermatol.

    (2000)
  • CurrieB.J. et al.

    First documentation of in vivo and in vitro ivermectin resistance in Sarcoptes scabiei

    Clin. Infect. Dis.

    (2004)
  • ArlianL.G. et al.

    Resistance and immune response in scabies-infested hosts immunized with Dermatophagoides mites

    Am. J. Trop. Med. Hyg.

    (1995)
  • MellanbyK.

    The development of symptoms, parasitic infection and immunity in human scabies

    Adv. Parasitol.

    (1944)
  • DougallA. et al.

    Identification and characterization of Sarcoptes scabiei and Dermatophagoides pteronyssinus glutathione S-transferases: implication as a potential major allergen in crusted scabies

    Am. J. Trop. Med. Hyg.

    (2005)
  • HarumalP. et al.

    Identification of a homologue of a house dust mite allergen in a cDNA library from Sarcoptes scabiei var hominis and evaluation of its vaccine potential in a rabbit/S. scabiei var. canis model

    Am. J. Trop. Med. Hyg.

    (2003)
  • HoltD.C. et al.

    Mechanisms for a novel immune evasion strategy in the scabies mite sarcoptes scabiei: a multigene family of inactivated serine proteases

    J. Invest. Dermatol.

    (2003)
  • DeperthesD.

    Phage display substrate: a blind method for determining protease specificity

    Biol. Chem.

    (2002)
  • Cited by (0)

    K.F., C.G.L. and J.A.I. contributed equally to this work.

    View full text