# Identification of new ribosylation-dependent immunoregulatory factors

> **NIH NIH R21** · STATE UNIVERSITY OF NEW YORK AT BUFFALO · 2020 · $199,375

## Abstract

Abstract
The evolution of multiple antibiotic-resistant pathogens for which treatment moieties are limited or absent makes
it essential to identify exploitable IMMUNE PATHWAYS to improve the efficacy of vaccines against infectious
agents. LT-IIb, a type II heat-labile enterotoxin (HLT), is a potent mucosal and systemic adjuvant that augments
antigen (Ag)-specific immune responses and elicits protection against pathogenic challenge in several mouse
immunization models. Enhancement of Ag-specific immune responses by LT-IIb, a bacterial ADP-ribsosylating
toxin, is regulated, in part, by its capacity to induce expression of IL-6, IL-1β, and several other cytokines by
immune cells. Current dogma is that the immunomodulatory properties of LT-IIb are mediated by the toxin’s
capacity to ADP-ribosylate the GSα regulatory subunit of the trimeric GSαβγ complex, which constitutively activates
adenylate cyclase (AD) in cells, eliciting a dramatic elevation in the intracellular concentration of cAMP, a strong
secondary signaling molecule. Preliminary experiments from our lab, however, CONTRADICT that dogmatic
model. We show that: (i) forskolin, an agent that strongly activates AD without requiring ADP-ribosylation of GSα,
fails to augment Ag-specific immune responses and does not induce IL-6 or IL-1β in immune cells; (ii)
pharmacological agents that inhibit AD and the GS trimer have no effect on elaboration of IL-6 by immune cells;
and (iii) LT-IIb(E59K/E110K)], an ADP-ribosylase-deficient mutant HLT, neither enhances immune responses
to co-administered Ag nor induces production of cytokines in immune cells. Thus, the capacity of LT-IIb to
mediate responses in immune cells requires an intact ADP-ribosylase activity, but does not require GSα , AD, or
cellular cAMP. Eukaryotic cells express ADP-ribosylases that modify proteins to regulate non-immune
pathways. We surmise that LT-IIb is mimicking an endogenous enzyme that modulates responses in immune
cells. Our HYPOTHESIS is that the immune responses augmented by LT-IIb are regulated by the toxin’s
capacity to ADP-ribosylate one or more proteins operating within an, as yet, undescribed
immunoregulatory pathway in B cells, macrophages, and other types of immune cells. To isolate the
protein(s) in murine B cells and macrophages that are modified by LT-IIb, we will employ LT-IIb as a
MOLECULAR PROBE and a recombinant polypeptide with strong binding for ADP-ribosylated proteins as an
affinity agent. Modified proteins obtained from cell lysates using this affinity agent will be identified using a
sophisticated, in-house-developed, high-resolution proteomics technology that is NOT BROADLY AVAILABLE
and designed to identify proteins in EXCEPTIONALLY LOW CONCENTRATIONS and WITHIN COMPLEX
MIXTURES. In a complementary set of experiments, human proteins modified by LT-IIb will be identified using
a ‘total human proteome’ microarray. These EXPLORATORY experiments will open new avenues of research
into the roles of ADP-ribosy...

## Key facts

- **NIH application ID:** 9869666
- **Project number:** 1R21AI148737-01
- **Recipient organization:** STATE UNIVERSITY OF NEW YORK AT BUFFALO
- **Principal Investigator:** Terry D. Connell
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $199,375
- **Award type:** 1
- **Project period:** 2020-03-20 → 2022-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9869666

## Citation

> US National Institutes of Health, RePORTER application 9869666, Identification of new ribosylation-dependent immunoregulatory factors (1R21AI148737-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9869666. Licensed CC0.

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