Glycoengineered Antibodies and Fluorogenic Sulfatase Probes
- Author(s): Smith, Elizabeth Lauren
- Advisor(s): Bertozzi, Carolyn R
- et al.
Sulfatases hydrolyze sulfate esters and are critical for regulating the sulfation state of biological systems. The active site of type I sulfatases contains an aldehyde-bearing formylglycine (fGly) residue required for catalytic activity; fGly is installed after the formylglycine-generating enzyme (FGE) recognizes the consensus CxPxR sequence and oxidizes the cysteine to fGly.
Our lab developed an approach to site-selectively modify proteins by installing the CxPxR motif onto proteins. The technique, called the aldehyde tag, involves cloning a variation of the consensus sequence, such as LCTPSR, into genes. Subsequently, fGly is incorporated site-specifically onto proteins of interest following conversion of Cys to fGly by FGE. The introduced aldehyde can then be used for protein modification using aldehyde selective chemistries.
Chapter One provides an overview of the aldehyde tag technique and the various technologies advanced by this protein modification method. Chapter Two reports the development of a platform to chemoselectively and site-specifically modify the crystallizable fragment (Fc) of IgG1. The natural Fc N-glycosylation sequon was replaced with a small aldehyde tag. Successful installation of fGly within the structured region required in vitro treatment with M. tuberculosis FGE; this internal aldehyde can then be chemically elaborated with various moieties to produce modified Fcs.
Chapter Three describes the synthesis of oxime-linked Fc glycoconjugates. Aldehyde tagged Fc was conjugated to aminooxy N-acetylglucosamine followed by chemoenzymatic transfer of complex glycan oxazolines by a glycosynthase, and the generated oxime-linked glycoconjugates emulate the structure of the natively glycosylated Fc. Fc glycans contribute to the overall structure and modulate Fc receptor binding; however, the Fc domain typically exists as a mixture of different glycoforms. Using this platform, homogeneous Fc glycoforms can be readily produced from numerous glycans to elucidate the role specific glycans have on antibody effector function.
Finally, Chapter Four presents two new fluorogenic sulfatase-activated probes, 3-O-methylfluorescein-sulfate and resorufin-sulfate. Both probes were validated with commercial sulfatases and employed to reveal species-specific sulfatase activity in mycobacterial lysates using a gel-based assay. Sulfatases are genetically conserved across many mycobacterial pathogens; however, their mechanism and function are not fully understood. These enzyme-activated probes can help clarify substrate specificity of sulfatases.