A biosynthetic route for polysialylating proteins in Escherichia coli.

Polysialic acid (polySia) is a posttranslational modification found on only a handful of proteins in the central nervous and immune systems. The addition of polySia to therapeutic proteins improves pharmacokinetics and reduces immunogenicity. To date, polysialylation of therapeutic proteins has only been achieved in vitro by chemical or chemoenzymatic strategies. In this work, we develop a biosynthetic pathway for site-specific polysialylation of recombinant proteins in the cytoplasm of Escherichia coli. The pathway takes advantage of a bacterial cytoplasmic polypeptide-glycosyltransferase to establish a site-specific primer on the target protein. The glucose primer is extended by glycosyltransferases derived from lipooligosaccharide, lipopolysaccharide and capsular polysaccharide biosynthesis from different bacterial species to synthesize long chain polySia. We demonstrate the new biosynthetic route by modifying green fluorescent proteins and a therapeutic DARPin (designed ankyrin repeat protein).

Analysis of site-specific N-glycan remodeling in the endoplasmic reticulum and the Golgi.

The hallmark of N-linked protein glycosylation is the generation of diverse glycan structures in the secretory pathway. Dynamic, non-template-driven processes of N-glycan remodeling in the endoplasmic reticulum and the Golgi provide the cellular setting for structural diversity. We applied newly developed mass spectrometry-based analytics to quantify site-specific N-glycan remodeling of the model protein Pdi1p expressed in insect cells. Molecular dynamics simulation, mutational analysis, kinetic studies of in vitro processing events and glycan flux analysis supported the defining role of the protein in N-glycan processing.

Dissecting the mechanisms of tissue transglutaminase-induced cross-linking of alpha-synuclein: implications for the pathogenesis of Parkinson disease.

Tissue transglutaminase (tTG) has been implicated in the pathogenesis of Parkinson disease (PD). However, exactly how tTG modulates the structural and functional properties of alpha-synuclein (alpha-syn) and contributes to the pathogenesis of PD remains unknown. Using site-directed mutagenesis combined with detailed biophysical and mass spectrometry analyses, we sought to identify the exact residues involved in tTG-catalyzed cross-linking of wild-type alpha-syn and alpha-syn mutants associated with PD. To better understand the structural consequences of each cross-linking reaction, we determined the effect of tTG-catalyzed cross-linking on the oligomerization, fibrillization, and membrane binding of alpha-syn in vitro. Our findings show that tTG-catalyzed cross-linking of monomeric alpha-syn involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed cross-linked monomeric alpha-syn composed of either wild-type or Gln --> Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping by mass spectrometry. Using this approach, we identified the glutamine and lysine residues involved in tTG-catalyzed intramolecular cross-linking of alpha-syn. These studies demonstrate for the first time that Gln(79) and Gln(109) serve as the primary tTG reactive sites. Mutating both residues to asparagine abolishes tTG-catalyzed cross-linking of alpha-syn and tTG-induced inhibition of alpha-syn fibrillization in vitro. To further elucidate the sequence and structural basis underlying these effects, we identified the lysine residues that form isopeptide bonds with Gln(79) and Gln(109). This study provides mechanistic insight into the sequence and structural basis of the inhibitory effects of tTG on alpha-syn fibrillogenesis in vivo, and it sheds light on the potential role of tTG cross-linking on modulating the physiological and pathogenic properties of alpha-syn.

Sphingomyelin synthase 1 suppresses ceramide production and apoptosis post-photodamage.

The role of sphingomyelin synthase 1 (SMS1), the Golgi membrane isoform of the enzyme, in ceramide metabolism and apoptosis after photodamage with the photosensitizer Pc 4 (PDT) is unclear. In the present study, using electrospray ionization/double mass spectrometry, we show that in Jurkat cells overexpressing SMS1, increases in ceramides were lower than in empty-vector transfectants post-PDT. Similarly, the responses of dihydroceramides and dihydrosphingosine, precursors of ceramide in the de novo synthetic pathway, were attenuated in SMS1-overexpressor after photodamage, suggesting the involvement of the de novo pathway. Overexpression of SMS1 was associated with differential regulation of sphingomyelin levels, as well as with the reduced inhibition of the enzyme post-treatment. Concomitant with the suppressed ceramide response, PDT-induced DEVDase activation was substantially reduced in SMS1-overexpressors. The data show that overexpression of SMS1 is associated with suppressed ceramide response and apoptotic resistance after photodamage.