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A suite of tools for the study of tryptophan C-mannosylation

ABSTRACT: Tryptophan C-mannosylation is a co-translational modification unique to the metazoans and apicomplexan parasites. It is the only example of protein C-glycosylation reported to date and appears to play a role in protein folding, trafficking and/or stability. Our knowledge of the prevalence of this modification and its role in biology is limited, and hampered by a lack of tools for studying the modification. Here, we present a simple microbial system for the production of proteins with and without the modification, to facilitate characterisation of the effects of tryptophan mannosylation on protein stability and function. This system facilitates exploration of the C-mannosyltransferase substrate preference and catalytic machinery. The ability to readily produce proteins with this modification facilitated the generation and rigours characterisation of monoclonal antibodies capable of detecting C-mannosyl tryptophan. With these antibodies, and a robust expression system, we were able to identify the first small molecule capable of inhibiting C-mannosyltransferases: a susbtrate-mimicking peptide that is active in cells. Together, these tools provide a solid foundation for interrogating the role of C-mannosyl tryptophan in myriad biological systems.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Nichollas Scott

LAB HEAD: Ethan Goddard-Borger

PROVIDER: PXD018401 | Pride | 2020-11-16

REPOSITORIES: Pride

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Project description:Type 1 diabetes mellitus (T1DM) results from immune mediated destruction of pancreatic beta cells. However, clinical and immunologic phenotypes of T1DM are variable. Several auto-antibodies including GADA, IA-2A, and ZnT8A, were identified in T1DM, but the prevalence of these auto-antibodies varied for a broad spectrum of T1DM. Here, we systemically profiled auto-antibodies from serum samples of 16 T1DM, 16 type 2 diabetes (T2DM) patients, and 27 healthy controls with normal glucose tolerance (NGT) using protein microarrays containing 9,480 proteins. Among 9,480 different proteins on the array, we identified novel auto-antibody candidates (EEF1A1-AAb and UBE2L3-AAb) by M-test coupled with PLS-DA. These auto-antibodies were highly present in T1DM than controls and detected in 40% of T1DM without GADA. Furthermore, these auto-antibodies might help to differentiate subtype of T1DM when combined with GADA. These novel auto-antibodies provide new diagnostic information of T1DM, as well as new insights into the pathogenesis of T1DM. Auto-antibodies from serum samples were profiled using a high-density, fluorescence-based protein microarray containing duplicate spots of 9,480 human proteins derived from the Ultimate ORF collection The cohort of patients and controls consisted of 16 T1DM, 16 T2DM patients, and 27 healthy controls with NGT. This cohort was used to screen candidate auto-antibodies using protein microarrays (ProtoArray platform version 5.0, Invitrogen Corp., Carlsbad, CA). Serum samples were drawn from T1DM patients who have 1) fasting C-peptide level <0.3 nmol/L or serum C-peptide <0.6 nmol/L after glucagon loading, 2) initiation of insulin treatment within six months after diagnosis, and 3) duration of diabetes M-bM-^IM-$12 months. Mean age of T1DM in the first cohort was 42 M-BM-1 16 years. The control serum samples were obtained from T2DM patients who were treated only with oral anti-diabetic drug at least 5 years and from NGTs who had no history of diabetes, no first-degree relatives with diabetes, a fasting plasma glucose concentration of <6.1 mmol/l, and a HbA1c value of <5.8%.

			Action	DRS
	CeUNC5.zip	Other
	EPOR_lysargiNase.zip	Other
	IL12B.zip	Other
	IL12R.zip	Other
	Mindin.zip	Other

Dataset Information

A suite of tools for the study of tryptophan C-mannosylation

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