Heparan sulphate binding controls in vivo half-life of the HpARI protein family
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eLife assessment
This valuable study uses in vitro and in vivo methods to identify HpARI proteins from H. polygyrus as modulators of the host immune system. Based on comprehensive approaches for investigating differential roles of HpARI proteins, the data are solid, but there are some concerns whether the claims are fully validate. This paper is relevant to those who investigate host-pathogen interactions at the systems and molecular levels.
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Abstract
The parasitic nematode Heligmosomoides polygyrus bakeri secretes the HpARI family, which bind to IL-33, either suppressing (HpARI1 and HpARI2) or enhancing (HpARI3) responses to the cytokine. We previously showed that HpARI2 also bound to DNA via its first Complement Control Protein (CCP1) domain. Here, we find that HpARI1 can also bind DNA, while HpARI3 cannot. Through the production of HpARI2/HpARI3 CCP1 domain-swapped chimeras, DNA-binding ability can be transferred, and correlates with in vivo half-life of administered proteins. We found that HpARI1 and HpARI2 (but not HpARI3) also binds to the extracellular matrix component heparan sulphate (HS), and structural modelling showed a basic charged patch in the CCP1 domain of HpARI1 and HpARI2 (but not HpARI3) which could facilitate these interactions. Finally, a mutant of HpARI2 was produced which lacked DNA and HS binding, and was also shown to have a short half-life in vivo. Therefore, we propose that during infection the suppressive HpARI1 and HpARI2 proteins have long-lasting effects, and may be retained at the infection sites via DNA and/or extracellular matrix interactions. Conversely, HpARI3 may have a shorter half-life of effects where deposited, but could diffuse to distal sites.
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eLife assessment
This valuable study uses in vitro and in vivo methods to identify HpARI proteins from H. polygyrus as modulators of the host immune system. Based on comprehensive approaches for investigating differential roles of HpARI proteins, the data are solid, but there are some concerns whether the claims are fully validate. This paper is relevant to those who investigate host-pathogen interactions at the systems and molecular levels.
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Reviewer #1 (Public Review):
Summary:
Colomb et al have further explored the mechanisms of action of a family of three immunodulatory proteins produced by the murine gastrointestinal nematode parasite Heligmosomoides polygyrus bakeri. The family of HpARI proteins binds to the alarmin interleukin 33 and depending on family members, exhibits differential activities, either suppressive or enhancing. The present work extends previous studies by this group showing the binding of DNA by members of this family through a complement control protein (CCP1) domain. Moreover, they identify two members of the family that bind via this domain in a non-specific manner to the extracellular matrix molecule heparan sulphate through a basic charged patch in CCP1. The authors thus propose that binding to DNA or heparan sulphate extends the suppressive …
Reviewer #1 (Public Review):
Summary:
Colomb et al have further explored the mechanisms of action of a family of three immunodulatory proteins produced by the murine gastrointestinal nematode parasite Heligmosomoides polygyrus bakeri. The family of HpARI proteins binds to the alarmin interleukin 33 and depending on family members, exhibits differential activities, either suppressive or enhancing. The present work extends previous studies by this group showing the binding of DNA by members of this family through a complement control protein (CCP1) domain. Moreover, they identify two members of the family that bind via this domain in a non-specific manner to the extracellular matrix molecule heparan sulphate through a basic charged patch in CCP1. The authors thus propose that binding to DNA or heparan sulphate extends the suppressive action of these two parasite molecules, whereas the third family member does not bind and consequently has a shorter half-life and may function via diffusion.
Strengths:
A strength of the work is the multifaceted approach to examining and testing their hypotheses, using a well-established and well-defined family of immunomodulatory molecules using multiple approaches including an in vivo setting.
Weaknesses:
There are a few weaknesses of the approach. Perhaps some discussion and speculation as to how these three family members might operate in concert during Heligmosomoides polygyrus bakeri infection would help place the biology of these molecules in context for the reader, e.g. when and where they are produced.
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Reviewer #2 (Public Review):
Summary:
Colomb et al. investigated here the heparin-binding activity of the HpARI family proteins from H. polygyrus. HpARIs bind to IL-33, a pleiotropic cytokine, and modulate its activities. HpARI1/2 has suppressive functions, while HpARI3 can enhance the interaction between IL-33 and its receptor. This study builds upon their previous observation that HpARI2 binds DNA via its CCP1 domain. Here, the authors tested the CCP1 domain of HpARIs in binding heparan sulfate, an important component of the extracellular matrix, and found that 1/2 bind heparan, but 3 cannot, which is related to their half-lives in vivo.
Strengths:
The authors use a comprehensive multidisciplinary approach to assess the binding and their effects in vivo, coupled with molecular modeling.
Weaknesses:
(1) Figure 1C should include Western.
Reviewer #2 (Public Review):
Summary:
Colomb et al. investigated here the heparin-binding activity of the HpARI family proteins from H. polygyrus. HpARIs bind to IL-33, a pleiotropic cytokine, and modulate its activities. HpARI1/2 has suppressive functions, while HpARI3 can enhance the interaction between IL-33 and its receptor. This study builds upon their previous observation that HpARI2 binds DNA via its CCP1 domain. Here, the authors tested the CCP1 domain of HpARIs in binding heparan sulfate, an important component of the extracellular matrix, and found that 1/2 bind heparan, but 3 cannot, which is related to their half-lives in vivo.
Strengths:
The authors use a comprehensive multidisciplinary approach to assess the binding and their effects in vivo, coupled with molecular modeling.
Weaknesses:
(1) Figure 1C should include Western.
(2) Figure 1E: Why does HpARI1 stop binding DNA at 50%?
(3) ITC binding experiment with HpARI1?Also, the ITC results from HpARI2 do not seem to saturate, thus it is difficult to really determine the affinity.
(4) It would be helpful to add docking results from HpARI1.
(5) Some conclusions are speculative and need to remain in the Discussion. e.g.:
a) That HpARI3 may be able to diffuse farther
b) That DNA/HS may trap HpARI1/2 at the infection site. -
