Viswanatham Katta

In Vitro Methionine Oxidation of Recombinant Human Leptin

AbstractPurpose. To investigate the role and importance of the four methionines in recombinant human leptin, and the effect of methionine oxidation in leptin structural stability and biological activity. Methods. Oxidized leptin derivatives were prepared in the presence of H2O2 and t-butylhydroperoxide, separated by RP-HPLC, and characterized by peptide mapping and LC/MS. Their biophysical and biological properties were studied. Results. Six major species of oxidized leptins were detected: two mono-oxidized, one di-oxidized, two tri-oxidized, and one tetra-oxidized. Further oxidation at cystine disulfide was also detected. Kinetic analysis indicated that oxidation at Met1 and Met69 proceeded first and independently. In 48 mM t-butylhydroperoxide, the pseudo first-order rate constants, k1 and k69, were 1.5 × 10−3 and 2.3 × 10−4 min−1. No change in the secondary or tertiary structure was detected for Met1 mono-oxidized and Met1, Met69 di-oxidized leptins. The Met1 mono-oxidized leptin retained full potency as compared to native leptin. A slight decrease of thermostability and a significant loss of the in vitro bioactivity were observed for Met1, Met69 di-oxidized leptin. Both Met55 and Met137 were not oxidized in t-butylhydroperoxide but only in H2O2. They appeared to be much less accessible to oxidation and might interact with the hydrophobic core structure of the leptin molecule. Conclusions. The oxidation of leptin occurred in the order of Met1 > Met69 >> Met55 ≈ Met137, and the importance for maintaining leptin structural integrity was Met55 ≈ Met137 >> Met69≈ Met1. Met69, but not Met1, plays a critical role in the protein stability and activity.

The Intermolecular Disulfide Bridge of Human Glial Cell Line-Derived Neurotrophic Factor: Its Selective Reduction and Biological Activity of the Modified Protein

Recombinant human glial cell line-derived neurotrophic factor has been implicated to have therapeutic potential in the treatment of neurodegenerative diseases. The mature protein is a single polypeptide of 134 amino acid residues and functions as a disulfide-linked dimer. Reduction of the protein with dithiothreitol at pH 7.0 and in the absence of denaturant showed that the single intermolecular cystine bridge was reduced preferentially. Direct alkylation of the generated free sulfhydryl group using iodoacetamide or iodoacetate without denaturant was incomplete. Unfolding the protein in 6 M guanidine hydrochloride prior to the modification showed rapid disulfide scrambling. However, the sulfhydryl-modifying reagent N-ethylmaleimide was able to label quantitatively the free cysteinyl residue in the absence of any added chaotropic agent. By a combination of peptide mapping, Edman degradation, and mass spectrometric analysis, the labeled residue was identified to be Cys101, hence verifying the location of the intermolecular disulfide bond. The modified protein behaved as a noncovalent dimer when chromatographed through a Superdex 75 column under nondenaturing conditions and was comparable in biological activity to an unmodified control sample. The results therefore indicate that the intermolecular disulfide bridge of the protein is not essential for its biological function.

Peptide map procedure using immobilized protease cartridges in tandem for disulfide linkage identification of neu differentiation factor epidermal growth factor domain

Immobilized proteolytic enzyme cartridges were used to rapidly digest neu differentiation factor EGF domain in order to obtain improved peptide maps useful for assignment of disulfide linkages. The procedure described here involves an on-line digestion of proteins using immobilized trypsin and endoproteinase Glu-C cartridges connected in series, followed by on-line RP-HPLC separation of the peptides. The entire process can be automated using a commercially available workstation; and the total time required for both proteolytic digestion and the HPLC separation can be shortened to within 1 h. Using these immobilized columns, we demonstrated that disulfide structure assignment of the EGF domains of recombinant human neu differentiation factor can be performed by isolation of individual disulfide-containing peptides followed by assignment of disulfide linkages with prompt fragmentation of peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The use of immobilized protease cartridges in tandem eliminates undesirable digestion artifacts associated with longer digestion time and higher protease-to-substrate ratio and results in the development of a reproducible and high quality peptide map.

Development and evaluation of on-line nanoliter flow analysis of protein digests by pneumatic-splitter electrospray liquid chromatography mass spectrometry

We have developed a novel approach to characterize protein digests by pneumatic-splitter electrospray ionization liquid chromatography mass spectrometry (PSESI-LCMS). This technique involves an interfacing of a pneumatic splitter that can dynamically generate and control a steady split flow rate of nanoliters per minute. An average PSESI-LCMS analysis, depending on the desired split ratio, consumes less than 200 femtomoles of sample. A tryptic digest map of a glycoprotein, fetuin, and a Lys-C digest map of a recombinant neurotrophin protein, neurotrophin-3, are reported here for this investigation.

Human neurotrophin-3: a one-step peptide mapping method and complete disulfide characterization of the recombinant protein.

Human neurotrophin-3 (NT-3) is a member of the nerve growth factor (NGF) family of neurotrophic factors, and the recombinant protein is being developed as a therapeutic for neurodegenerative diseases. The final product purity and lot-to-lot variation are monitored routinely by peptide mapping. However, only the N-terminal region of NT-3 was susceptible to proteolysis under native conditions. Complete digestion required that the protein be chemically modified by reduction and S-alkylation prior to proteolysis. Complete proteolytic degradation of the protein was achieved simply by an intial denaturation of NT-3 in 6 M guanidinium chloride (pH 6) for 2 hr at 37°C, followed by a tenfold dilution with the digestion buffer (0.1 M Tris-HCl, 1 mM CaCl2 at pH 7.0) and immediate addition of chymotrypsin at 1% by weight. Direct comparison of the peptide map with an identical aliquot that had been reduced and alkylated also allowed the establishment of the cystine linkages present in NT-3: Cys14 to Cys79, Cys57 to Cys108, and Cys67 to Cys110. This disulfide structure is homologous to the NGF family of neurotrophic factors.

Selective reduction of the intermolecular disulfide bridge in human glial cell line-derived neurotrophic factor using tris-(2-carboxyethyl) phosphine

Publisher Summary Glial cell line-derived neurotrophic factor (GDNF) is one of the more recently identified neurotrophic factors, first purified from the conditioned medium of a rat glial cell line (B49). Because of its ability in supporting the growth of midbrain dopaminergic neurons in vitro , GDNF has been implicated to have therapeutic potential in the treatment of Parkinsons disease. Mature GDNF is a single polypeptide with 134 amino acid residues, containing seven cysteines and functions as a glycosylated, disulfide linked dimer. Examination of the proteins primary structure suggests that GDNF is a distant member of the transforming growth factor-fi (TGF-fi) superfamily of growth factors. The recombinant protein has been expressed in Escherichia coli and is currently being developed as a candidate for human therapeutic use. Because of its complex structure, the protein was refractory to extensive proteolytic degradation under native conditions. This chapter discusses the issue of disulfide assignment by subjecting GDNF to partial reduction using the chemical reagent tris-(2-carboxyethyl)phosphine (TCEP) in 0.17 M acetic acid at pH 2.5 and the structural characterization of the single reduced disulfide bond is reviewed.

PROTEIN OXIDATION BY MYELOPEROXIDASE (MPO) IN VITRO IS DISTINGUISHABLE FROM OXIDATION BY REAGENT HOCI. 337

PROTEIN OXIDATION BY MYELOPEROXIDASE (MPO) IN VITRO IS DISTINGUISHABLE FROM OXIDATION BY REAGENT HOCI. 337

Asparagine rearrangements in two isoforms of erythropoietin receptor

Publisher Summary This chapter discusses the asparagine rearrangements in two isoforms of erythropoietin receptor (EPOr). The two isoforms of the EPO receptor—EPOr-1 and EPOr-2—differ in charge from each other because of a modification in EPOr-2 of Asn-163 to isoaspartate. This modification demonstrated that a small portion of EPOr-1 contained a cyclic imide. Asparagine, followed by a glycyl residue, may form a succinimide or cyclic imide at a high rate. EPOr contains one Asn-Gly combination at residues 163–164. To discern if modification of Asn-163 was the difference between these two isoforms, both EPOr-1 and EPOr-2 were examined by hydroxylamine cleavage using conditions that would cleave a cyclic imide only. A portion of EPOr-1 was cleaved, but EPOr-2 alone was not cleaved, indicating the presence of a cyclic imide in EPOr-1. Tryptic peptide mapping on reverse phase high-performance liquid chromatography (HPLC) was followed by matrix assisted laser desorption/ ionization mass spectrometry (MALDI/MS) of the peptides. A peptide from each isoform with a mass consistent with the mass of residues 155–170 was identified. N-terminal sequencing of these two peptides revealed that the major part of EPOr-1 had an Asn at residue 163. This residue in EPOr-2 was not detected by N-terminal sequencing, indicating that a modification of residue 163 had occurred that would block further sequencing. Rearrangement of asparagine to an isoaspartate would be such a modification.