Cytoplasmic glycoengineering enables biosynthesis of nanoscale glycoprotein assemblies.

Glycosylation of proteins profoundly impacts their physical and biological properties. Yet our ability to engineer novel glycoprotein structures remains limited. Established bacterial glycoengineering platforms require secretion of the acceptor protein to the periplasmic space and preassembly of the oligosaccharide substrate as a lipid-linked precursor, limiting access to protein and glycan substrates respectively. Here, we circumvent these bottlenecks by developing a facile glycoengineering platform that operates in the bacterial cytoplasm. The Glycoli platform leverages a recently discovered site-specific polypeptide glycosyltransferase together with variable glycosyltransferase modules to synthesize defined glycans, of bacterial or mammalian origin, directly onto recombinant proteins in the E. coli cytoplasm. We exploit the cytoplasmic localization of this glycoengineering platform to generate a variety of multivalent glycostructures, including self-assembling nanomaterials bearing hundreds of copies of the glycan epitope. This work establishes cytoplasmic glycoengineering as a powerful platform for producing glycoprotein structures with diverse future biomedical applications.

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).

Recombinant expression of hydroxylated human collagen in Escherichia coli.

Collagen is the most abundant protein in the human body and thereby a structural protein of considerable biotechnological interest. The complex maturation process of collagen, including essential post-translational modifications such as prolyl and lysyl hydroxylation, has precluded large-scale production of recombinant collagen featuring the biophysical properties of endogenous collagen. The characterization of new prolyl and lysyl hydroxylase genes encoded by the giant virus mimivirus reveals a method for production of hydroxylated collagen. The coexpression of a human collagen type III construct together with mimivirus prolyl and lysyl hydroxylases in Escherichia coli yielded up to 90 mg of hydroxylated collagen per liter culture. The respective levels of prolyl and lysyl hydroxylation reaching 25 % and 26 % were similar to the hydroxylation levels of native human collagen type III. The distribution of hydroxyproline and hydroxylysine along recombinant collagen was also similar to that of native collagen as determined by mass spectrometric analysis of tryptic peptides. The triple helix signature of recombinant hydroxylated collagen was confirmed by circular dichroism, which also showed that hydroxylation increased the thermal stability of the recombinant collagen construct. Recombinant hydroxylated collagen produced in E. coli supported the growth of human umbilical endothelial cells, underlining the biocompatibility of the recombinant protein as extracellular matrix. The high yield of recombinant protein expression and the extensive level of prolyl and lysyl hydroxylation achieved indicate that recombinant hydroxylated collagen can be produced at large scale for biomaterials engineering in the context of biomedical applications.

Identification of domains and amino acids essential to the collagen galactosyltransferase activity of GLT25D1.

Collagen is modified by hydroxylation and glycosylation of hydroxylysine residues. This glycosylation is initiated by the ß1,O galactosyltransferases GLT25D1 and GLT25D2. The structurally similar protein cerebral endothelial cell adhesion molecule CEECAM1 was previously reported to be inactive when assayed for collagen glycosyltransferase activity. To address the cause of the absent galactosyltransferase activity, we have generated several chimeric constructs between the active human GLT25D1 and inactive human CEECAM1 proteins. The assay of these chimeric constructs pointed to a short central region and a large C-terminal region of CEECAM1 leading to the loss of collagen galactosyltransferase activity. Examination of the three DXD motifs of the active GLT25D1 by site-directed mutagenesis confirmed the importance of the first (amino acids 166-168) and second motif (amino acids 461-463) for enzymatic activity, whereas the third one was dispensable. Since the second DXD motif is incomplete in CEECAM1, we have restored the motif by introducing the substitution S461D. This change did not restore the activity of the C-terminal region, thereby showing that additional amino acids were required in this C-terminal region to confer enzymatic activity. Finally, we have introduced the substitution Q471R-V472M-N473Q-P474V in the CEECAM1-C-terminal construct, which is found in most animal GLT25D1 and GLT25D2 isoforms but not in CEECAM1. This substitution was shown to partially restore collagen galactosyltransferase activity, underlining its importance for catalytic activity in the C-terminal domain. Because multiple mutations in different regions of CEECAM1 contribute to the lack of galactosyltransferase activity, we deduced that CEECAM1 is functionally different from the related GLT25D1 protein.

Core glycosylation of collagen is initiated by two beta(1-O)galactosyltransferases.

Collagen is a trimer of three left-handed alpha chains representing repeats of the motif Gly-X-Y, where (hydroxy)proline and (hydroxy)lysine residues are often found at positions X and Y. Selected hydroxylysines are further modified by the addition of galactose and glucose-galactose units. Collagen glycosylation takes place in the endoplasmic reticulum before triple-helix formation and is mediated by beta(1-O)galactosyl- and alpha(1-2)glucosyltransferase enzymes. We have identified two collagen galactosyltransferases using affinity chromatography and tandem mass spectrometry protein sequencing. The two collagen beta(1-O)galactosyltransferases corresponded to the GLT25D1 and GLT25D2 proteins. Recombinant GLT25D1 and GLT25D2 enzymes showed a strong galactosyltransferase activity toward various types of collagen and toward the serum mannose-binding lectin MBL, which contains a collagen domain. Amino acid analysis of the products of GLT25D1 and GLT25D2 reactions confirmed the transfer of galactose to hydroxylysine residues. The GLT25D1 gene is constitutively expressed in human tissues, whereas the GLT25D2 gene is expressed only at low levels in the nervous system. The GLT25D1 and GLT25D2 enzymes are similar to CEECAM1, to which we could not attribute any collagen galactosyltransferase activity. The GLT25D1 and GLT25D2 genes now allow addressing of the biological significance of collagen glycosylation and the importance of this posttranslational modification in the etiology of connective tissue disorders.