Dingyi Wen

Identification of a Palmitic Acid-modified Form of Human Sonic hedgehog

During hedgehog biosynthesis, autocatalytic processing produces a lipid-modified amino-terminal fragment (residues 24–197 in the human Sonic hedgehog sequence) that is responsible for all known hedgehog signaling activity and that is highly conserved evolutionarily. Published in vitro biochemical studies using Drosophila hedgehog identified the membrane anchor as a cholesterol, and localized the site of attachment to the COOH terminus of the fragment. We have expressed full-length human Sonic hedgehog in insect and in mammalian cells and determined by mass spectrometry that, in addition to cholesterol, the human hedgehog protein is palmitoylated. Peptide mapping and sequencing data indicate that the palmitoyl group is attached to the NH2 terminus of the protein on the α-amino group of Cys-24. Cell-free palmitoylation studies demonstrate that radioactive palmitic acid is readily incorporated into wild type Sonic hedgehog, but not into variant forms lacking the Cys-24 attachment site. The lipid-tethered forms of hedgehog showed about a 30-fold increase in potency over unmodified soluble hedgehog in a cell- based (C3H10T1/2 alkaline phosphatase induction) assay, suggesting that the lipid tether plays an important role in hedgehog function. The observation that an extracellular protein such as Shh is palmitoylated is highly unusual and further adds to the complex nature of this protein.

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.

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 tRNAAsn 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.

Growth factor induction of cripto-1 shedding by glycosylphosphatidylinositol-phospholipase D and enhancement of endothelial cell migration

Cripto-1 (CR-1) is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that has been shown to play an important role in embryogenesis and cellular transformation. CR-1 is reported to function as a membrane-bound co-receptor and as a soluble ligand. Although a number of studies implicate the role of CR-1 as a soluble ligand in tumor progression, it is unclear how transition from the membrane-bound to the soluble form is physiologically regulated and whether differences in biological activity exist between these forms. Here, we demonstrate that CR-1 protein is secreted from tumor cells into the conditioned medium after treatment with serum, epidermal growth factor, or lysophosphatidic acid, and this soluble form of CR-1 exhibits the ability to promote endothelial cell migration as a paracrine chemoattractant. On the other hand, membrane-bound CR-1 can stimulate endothelial cell sprouting through direct cell-cell interaction. Shedding of CR-1 occurs at the GPI-anchorage site by the activity of GPI-phospholipase D (GPI-PLD), because CR-1 shedding was suppressed by siRNA knockdown of GPI-PLD and enhanced by overexpression of GPI-PLD. These findings describe a novel molecular mechanism of CR-1 shedding, which may contribute to endothelial cell migration and possibly tumor angiogenesis.

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.

Expression of Sonic hedgehog-Fc fusion protein in Pichia pastoris. Identification and control of post-translational, chemical, and proteolytic modifications

We have investigated the suitability of Pichia pastoris as an expression system for the candidate therapeutic protein, Sonic hedgehog fused to an immunoglobulin Fc domain (Shh-Fc). Sonic hedgehog is a morphogen protein involved in the patterning of a wide range of tissues during animal embryogenesis. The presence of Sonic hedgehog and its receptor, Patched, in adult nervous tissue suggests possible applications for the protein in the treatment of neurodegenerative disease and injury. We have engineered the Shh-Fc fusion protein in order to improve binding affinity and increase systemic exposure in animals. N-terminal sequencing, peptide mapping, mass spectrometry, and other biochemical and biological methods were used to characterize the purified protein. These analyses revealed several unanticipated problems, including thiaproline modification of the N-terminal cysteine, cleavage by a Kex2-like protease at a site near the N-terminus, proteolysis at sites near the hinge, addition of a hexose in the CH3 domain of the Fc region, and several sites of methionine oxidation. Sequence modifications to the protein and changes in fermentation conditions resulted in increased potency and greater consistency of the product. The final product was shown to be biologically active in animal studies.

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.

Resolution of disulfide heterogeneity in Nogo receptor 1 fusion proteins by molecular engineering

NgR1 (Nogo‐66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgR1 have been used successfully to promote axon regeneration in animal models of spinal‐cord injury, raising interest in this protein as a potential therapeutic target. The LRR (leucine‐rich repeat) regions in NgR1 are flanked by N‐ and C‐terminal disulfide‐containing ‘cap’ domains (LRRNT and LRRCT respectively). In the present work we show that, although functionally active, the NgR1(310)‐Fc fusion protein contains mislinked and heterogeneous disulfide patterns in the LRRCT domain, and we report the generation of a series of variant molecules specifically designed to prevent this heterogeneity. Using these variants we explored the effects of modifying the NgR1 truncation site or the spacing between the NgR1 and Fc domains, or replacing cysteines within the NgR1 or IgG hinge regions. One variant, which incorporates replacements of Cys266 and Cys309 with alanine residues, completely eliminated disulfide scrambling while maintaining functional in vitro and in vivo efficacy. This modified NgR1‐Fc molecule represents a significantly improved candidate for further pharmaceutical development, and may serve as a useful model for the optimization of other IgG fusion proteins made from LRR proteins.

A Prodomain Fragment from the Proteolytic Activation of Growth Differentiation Factor 11 Remains Associated with the Mature Growth Factor and Keeps It Soluble

Growth differentiation factor 11 (GDF11), a member of the transforming growth factor β (TGF-β) family, plays diverse roles in mammalian development. It is synthesized as a large, inactive precursor protein containing a prodomain, pro-GDF11, and exists as a homodimer. Activation requires two proteolytic processing steps that release the prodomains and transform latent pro-GDF11 into active mature GDF11. In studying proteolytic activation in vitro, we discovered that a 6-kDa prodomain peptide containing residues 60-114, PDP60-114, remained associated with the mature growth factor. Whereas the full-length prodomain of GDF11 is a functional antagonist, PDP60-114 had no impact on activity. The specific activity of the GDF11/PDP60-114 complex (EC50 = 1 nM) in a SMAD2/3 reporter assay was identical to that of mature GDF11 alone. PDP60-114 improved the solubility of mature GDF11 at neutral pH. As the growth factor normally aggregates/precipitates at neutral pH, PDP60-114 can be used as a solubility-enhancing formulation. Expression of two engineered constructs with PDP60-114 genetically fused to the mature domain of GDF11 through a 2x or 3x G4S linker produced soluble monomeric products that could be dimerized through redox reactions. The construct with a 3x G4S linker retained 10% activity (EC50 = 10 nM), whereas the construct connected with a 2x G4S linker could only be activated (EC50 = 2 nM) by protease treatment. Complex formation with PDP60-114 represents a new strategy for stabilizing GDF11 in an active state that may translate to other members of the TGF-β family that form latent pro/mature domain complexes.

Determination of the Disulfide Structure of Murine Meteorin, a Neurotrophic Factor, by LC–MS and Electron Transfer Dissociation-High-Energy Collisional Dissociation Analysis of Proteolytic Fragments

Meteorin and Cometin (Meteorin-like) are secreted proteins belonging to a newly discovered growth factor family. Both proteins play important roles in neural development and may have potential as therapeutic targets or agents. Meteorin and Cometin are homologues and contain ten evolutionarily conserved Cys residues across a wide variety of species. However, the status of the Cys residues has remained unknown. Here, we have successfully determined the disulfide structure for murine Meteorin by LC-MS analysis of fragments generated by trypsin plus endoprotease-Asp-N. For proteolytic fragments linked by more than one disulfide bond, we used electron transfer dissociation (ETD) to partially dissociate disulfide bonds followed by high-energy collisional dissociation (HCD) to determine disulfide linkages. Our analysis revealed that the ten Cys residues in murine Meteorin form five disulfide bonds with Cys7 (C1) linked to Cys28 (C2), Cys59 (C3) to Cys95 (C4), Cys148 (C5) to Cys219 (C8), Cys151 (C6) to Cys243 (C9), and Cys161 (C7) to Cys266 (C10). Since the ten Cys residues are highly conserved in Meteorin and Cometin, it is likely that the disulfide linkages are also conserved. This disulfide structure information should facilitate structure-function relationship studies on this new class of neurotrophic factors and also assist in evaluation of their therapeutic potentials.