Research Article
Human and viral membrane–associated E3 ubiquitin ligases MARCH1 and MIR2 recognize different features of CD86 to downregulate surface expression

https://doi.org/10.1016/j.jbc.2021.100900Get rights and content
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Immune-stimulatory ligands, such as major histocompatibility complex molecules and the T-cell costimulatory ligand CD86, are central to productive immunity. Endogenous mammalian membrane-associated RING-CHs (MARCH) act on these and other targets to regulate antigen presentation and activation of adaptive immunity, whereas virus-encoded homologs target the same molecules to evade immune responses. Substrate specificity is encoded in or near the membrane-embedded domains of MARCHs and the proteins they regulate, but the exact sequences that distinguish substrates from nonsubstrates are poorly understood. Here, we examined the requirements for recognition of the costimulatory ligand CD86 by two different MARCH-family proteins, human MARCH1 and Kaposi's sarcoma herpesvirus modulator of immune recognition 2 (MIR2), using deep mutational scanning. We identified a highly specific recognition surface in the hydrophobic core of the CD86 transmembrane (TM) domain (TMD) that is required for recognition by MARCH1 and prominently features a proline at position 254. In contrast, MIR2 requires no specific sequences in the CD86 TMD but relies primarily on an aspartic acid at position 244 in the CD86 extracellular juxtamembrane region. Surprisingly, MIR2 recognized CD86 with a TMD composed entirely of valine, whereas many different single amino acid substitutions in the context of the native TM sequence conferred MIR2 resistance. These results show that the human and viral proteins evolved completely different recognition modes for the same substrate. That some TM sequences are incompatible with MIR2 activity, even when no specific recognition motif is required, suggests a more complicated mechanism of immune modulation via CD86 than was previously appreciated.

Keywords

membrane-associated E3 ubiquitin ligase
deep mutational scanning
protein–protein interactions
immune regulation
Kaposi's sarcoma virus
membrane protein

Abbreviations

DMS
deep mutational scanning
DOX
doxycycline
HLA
human leukocyte antigen
ICAM-1
intercellular adhesion molecule 1
IRES
internal ribosome entry site
JM
juxtamembrane
KSHV
Kaposi's sarcoma herpesvirus
MARCH
membrane-associated RING-CH
MFI
mean fluorescence intensity
MHC
major histocompatibility complex
MIR
modulator of immune recognition
PolyVal TMD
PolyValine TM domain
TM
transmembrane
TMD
transmembrane domain
UMI
unique molecular identifier

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Present address for Raphael Trenker: Cardiovascular Research Institute, University of California, San Francisco, California 94158, USA.