Development and analysis of alpha 1-antitrypsin neoglycoproteins: the impact of additional N-glycosylation sites on serum half-life.

Therapeutic efficacy of glycoproteins is affected by many factors, including molecular size and net charge; both are influenced by the presence and composition of glycan structures. Human alpha 1-antitrypsin (A1AT) was cloned and expressed in human embryonic kidney cells (HEK293) that are capable of mammalian glycosylation. Utilizing PCR-based site-directed mutagenesis, new A1AT variants were created with single, double, or triple additional N-glycosylation sites to the three naturally occurring N-glycosylation sites. Because of the supplementary N-glycans, the A1AT variants exhibited an increased molecular weight. Retention of inhibitory activity was shown via trypsin inhibitory assay. The A1AT variants were treated with PNGase F, and the resulting N-glycans were analyzed by MALDI-TOF mass spectrometry. The N-glycan profile of the recombinant A1AT variants was mostly composed of monofucosylated bi-, tri-, and tetraantennary complex-type N-glycans, with a tendency toward higher antennary structures compared to the wild-type. The relevance of N-glycosylation in A1AT for the circulatory serum half-life was demonstrated in CD1 mice. The A1AT neoglycoprotein with an additional N-glycosylation site at position N123 exhibited a 62% increase in serum half-life. Additionally, using a two-compartment model, the A1AT variants exhibited increased α-phase values, especially N123 (223%) and N201 (255%). The results suggest the recombinant A1AT neoglycoprotein as a serious alternative to A1AT derived from human plasma.

Construction and analysis of a novel peptide tag containing an unnatural N-glycosylation site.

The addition of N-glycans to clinically used proteins enhances their therapeutic features. Here we report the design of a novel peptide tag with an unnatural N-glycosylation site, which may increase the N-glycan content of generally any protein. The designed GlycoTags were attached to A1AT, EPO and AGP and constructs were expressed in HEK293 or CHO cells. Hereby we could prove that the attached unnatural N-glycosylation site is decorated with complex-type N-glycans and that the spacer as well as the C-terminal "tail" sequence are critical for the usage of the novel N-glycosylation site. This demonstrates that the novel GlycoTag is a convenient tool to provide proteins with extra N-glycan moieties by simply adding a peptide tag sequence as small as 22 amino acids.

Role of EAL-containing proteins in multicellular behavior of Salmonella enterica serovar Typhimurium.

GGDEF and EAL domain proteins are involved in turnover of the novel secondary messenger cyclic di(3'-->5')-guanylic acid (c-di-GMP) in many bacteria. The rdar morphotype, a multicellular behavior of Salmonella enterica serovar Typhimurium characterized by the expression of the extracellular matrix components cellulose and curli fimbriae is controlled by c-di-GMP. In this work the roles of the EAL and GGDEF-EAL domain proteins on rdar morphotype development were investigated. Knockout of four of 15 EAL and GGDEF-EAL domain proteins upregulated rdar morphotype expression and expression of CsgD, the central regulator of the rdar morphotype, and partially downregulated c-di-GMP concentrations. More-detailed analysis showed that the EAL domain protein STM4264 and the GGDEF-EAL domain protein STM1703, which highly downregulated the rdar morphotype, have overlapping yet distinct functions. Another subset of EAL and GGDEF-EAL domain proteins influenced multicellular behavior in liquid culture and flagellum-mediated motility. Consequently, this work has shown that several EAL and GGDEF-EAL domain proteins, which act as phosphodiesterases, play a determinative role in the expression level of multicellular behavior of Salmonella enterica serovar Typhimurium.