Discovery and investigation of O-xylosylation in engineered proteins containing a (GGGGS)n linker.

Protein engineering is a powerful tool for designing or modifying therapeutic proteins for enhanced efficacy, greater safety, reduced immunogenicity, and better delivery. GGGGS [(G4S)n] linkers are commonly used when engineering a protein, because of their flexibility and resistance to proteases. However, post-translational modifications (PTMs) can occur at the Ser residue in these linkers. Here, we report, for the first time, the occurrence of O-xylosylation at the serine residue in (G4S)n>2 linkers. The O-xylosylation was discovered as a result of molecular mass determination, peptide mapping analysis, and MS/MS sequencing. Our investigation showed that (i) O-xylosylation is a common PTM for (G4S)(n>2) linkers; (ii) GSG is the motif for O-xylosylation; and (iii) the total amount of xylosylation per linker increases as the number of GSG motifs in the linker increases. Our investigation has also shown that the O-xylosylation level is clone-dependent, to a certain degree, but the xylosylation level varies considerably among the proteins examined-from <2% to >25% per linker-likely depending on the accessibility to the sites by the xylosyltransferase. Our work demonstrates that potential therapeutic proteins containing (G4S)n linkers should be closely monitored for O-xylosylation in order to ensure that drugs are homogeneous and of high quality. The strategies for elimination and reduction of O-xylosylation were also examined and are discussed.

Multiple actions of systemic artemin in experimental neuropathy.

The clinical management of neuropathic pain is particularly challenging. Current therapies for neuropathic pain modulate nerve impulse propagation or synaptic transmission; these therapies are of limited benefit and have undesirable side effects. Injuries to peripheral nerves result in a host of pathophysiological changes associated with the sustained expression of abnormal pain. Here we show that systemic, intermittent administration of artemin produces dose- and time-related reversal of nerve injury-induced pain behavior, together with partial to complete normalization of multiple morphological and neurochemical features of the injury state. These effects of artemin were sustained for at least 28 days. Higher doses of artemin than those completely reversing experimental neuropathic pain did not elicit sensory or motor abnormalities. Our results indicate that the behavioral symptoms of neuropathic pain states can be treated successfully, and that partial to complete reversal of associated morphological and neurochemical changes is achievable with artemin.

Discovery and investigation of misincorporation of serine at asparagine positions in recombinant proteins expressed in Chinese hamster ovary cells.

Misincorporation of amino acids in proteins expressed in Escherichia coli has been well documented but not in proteins expressed in mammalian cells under normal recombinant protein production conditions. Here we report for the first time that Ser can be incorporated at Asn positions in proteins expressed in Chinese hamster ovary cells. This misincorporation was discovered as a result of intact mass measurement, peptide mapping analysis, and tandem mass spectroscopy sequencing. Our analyses showed that the substitution was not related to specific protein molecules or DNA codons and was not site-specific. We believe that the incorporation of Ser at sites coded for Asn was due to mischarging of tRNA(Asn) rather than to codon misreading. The rationale for substitution of Asn by Ser and not by other amino acids is also discussed. Further investigation indicated that the substitution was due to the starvation for Asn in the cell culture medium and that the substitution could be limited by using the Asn-rich feed. These observations demonstrate that the quality of expressed proteins should be closely monitored when altering cell culture conditions.

Characterization of trisulfide modification in antibodies.

Trisulfides are a posttranslational modification formed by the insertion of a sulfur atom into a disulfide bond. Although reports for trisulfides in proteins are limited, we find that they are a common modification in natural and recombinant antibodies of all immunoglobulin G (IgG) subtypes. Trisulfides were detected only in interchain linkages and were predominantly in the light-heavy linkages. Factors that lead to trisulfide formation and elimination and their impact on activity and stability were investigated. The peptide mapping methods developed for characterization and quantification of trisulfides should be applicable to any antibody and can be easily adapted for other types of proteins.

Picomole-level mapping of protein disulfides by mass spectrometry following partial reduction and alkylation.

We have deduced the disulfide bond linkage patterns, at very low protein levels (<0.5 nmol), in two cysteine-rich polypeptide domains using a new strategy involving partial reduction/alkylation of the protein, followed by peptide mapping and tanden mass spectrometry (MS/MS) sequencing on a nanoflow liquid chromatography-MS/MS system. The substrates for our work were the cysteine-rich ectodomain of human Fn14, a member of the tumor necrosis factor receptor family, and the IgV domain of murine TIM-1 (T-cell, Ig domain, and mucin domain-1). We have successfully determined the disulfide linkages for Fn14 and independently confirmed those of the IgV domain of TIM-1, whose crystal structure was published recently. The procedures that we describe here can be used to determine the disulfide structures for proteins with complex characteristics. They will also provide a means to obtain important information for structure-function studies and to ensure correct protein folding and batch-to-batch consistency in commercially produced recombinant proteins.

The CRIPTO/FRL-1/CRYPTIC (CFC) domain of human Cripto. Functional and structural insights through disulfide structure analysis.

The disulfide structure of the CRIPTO/FRL-1/CRYPTIC (CFC) domain of human Cripto protein was determined by a combination of enzymatic and chemical fragmentation, followed by chromatographic separation of the fragments, and characterization by mass spectrometry and N-terminal sequencing. These studies showed that Cys115 forms a disulfide bond with Cys133, Cys128 with Cys149, and Cys131 with Cys140. Protein database searching and molecular modeling revealed that the pattern of disulfide linkages in the CFC domain of Cripto is the same as that in PARS intercerebralis major Peptide C (PMP-C), a serine protease inhibitor, and that the EGF-CFC domains of Cripto are predicted to be structurally homologous to the EGF-VWFC domains of the C-terminal extracellular portions of Jagged 1 and Jagged 2. Biochemical studies of the interactions of ALK4 with the CFC domain of Cripto by fluorescence-activated cell sorter analysis indicate that the CFC domain binds to ALK4 independent of the EGF domain. A molecular model of the CFC domain of Cripto was constructed based on the nuclear magnetic resonance structure of PMP-C. This model reveals a hydrophobic patch in the domain opposite to the presumed ALK4 binding site. This hydrophobic patch may be functionally important for the formation of intra or intermolecular complexes.