Characterization by tandem mass spectrometry of structural modifications in proteins.

Tandem mass spectrometry can be used to solve a number of protein structural problems that are not amenable to conventional methods for amino acid sequencing. Typical problems that use this approach involve characterization of peptides with blocked amino termini or peptides that have been otherwise posttranslationally processed, such as, by phosphorylation or sulfation. The structure and homogeneity of synthetic peptides can also be evaluated. Since peptides can be selectively characterized in the presence of other peptides or contaminants, the need for extensive purification is reduced or eliminated.

gamma-Carboxyglutamic acids 36 and 40 do not contribute to human factor IX function.

The gamma-carboxyglutamic acid (Gla) domains of the vitamin K-dependent blood coagulation proteins contain 10 highly conserved Gla residues within the first 33 residues, but factor IX is unique in possessing 2 additional Gla residues at positions 36 and 40. To determine their importance, factor IX species lacking these Gla residues were isolated from heterologously expressed human factor IX. Using ion-exchange chromatography, peptide mapping, mass spectrometry, and N-terminal sequencing, we have purified and identified two partially carboxylated recombinant factor IX species; factor IX/gamma 40E is uncarboxylated at residue 40 and factor IX/gamma 36,40E is uncarboxylated at both residues 36 and 40. These species were compared with the fully gamma-carboxylated recombinant factor IX, unfractionated recombinant factor IX, and plasma-derived factor IX. As monitored by anti-factor IX:Ca (II)-specific antibodies and by the quenching of intrinsic fluorescence, all these factor IX species underwent the Ca(II)-induced conformational transition required for phospholipid membrane binding and bound equivalently to phospholipid vesicles composed of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine. Endothelial cell binding was also similar in all species, with half-maximal inhibition of the binding of 125I-labeled plasma-derived factor IX at concentrations of 2-6 nM. Functionally, factor IX/gamma 36,40E and factor IX/gamma 40E were similar to fully gamma-carboxylated recombinant factor IX and plasma-derived factor IX in their coagulant activity and in their ability to participate in the activation of factor X in the tenase complex both with synthetic phospholipid vesicles and activated platelets. However, Gla 36 and Gla 40 represent part of the epitope targeted by anti-factor IX:Mg(II)-specific antibodies because these antibodies bound factor IX preferentially to factor IX/gamma 36,40E and factor IX/gamma 40E. These results demonstrate that the gamma-carboxylation of glutamic acid residues 36 and 40 in human factor IX is not required for any function of factor IX examined.

Structural and functional characterization of B-domain deleted recombinant factor VIII.

A new high-purity recombinant factor VIII preparation has been developed for the treatment of hemophilia A. Structurally, this factor VIII preparation, B-domain deleted recombinant factor VIII (BDDrFVIII), differs from other recombinant and plasma-derived factor VIII preparations in that most of the B-domain has been deleted. To ensure that BDDrFVIII contains the requisite structural and functional features, it has been subjected to detailed biochemical and biophysical characterization in comparison to the plasma-derived form of factor VIII. Laboratory studies have shown that the primary, secondary, and tertiary structures of BDDrFVIII and the posttranslational modifications are similar to those of the [80 + 90]-kd form of plasma-derived factor VIII. In addition, BDDrFVIII has full biologic activity compared with full-length factor VIII preparations.

The rapid determination of erythrocyte porphyrins using reversed-phase high performance liquid chromatography.

The extraction, separation and quantification of free acid erythrocyte porphyrins are described. The porphyrins are extracted from whole blood using 3:1 ethyl acetate/acetic acid (v/v) and separated using the reversed-phase mode of high performance liquid chromatography (RPLC). The compounds are detected on-line spectrofluorometrically using an excitation wavelength of 404 nm and an emission cut-off filter of 550 nm. Excellent resolution of the porphyrin free acids is achieved in less than 6 min. The analytical recoveries for protoporphyrin IX and zinc protoporphyrin IX were greater than 90% with relative standard deviations for day-to-day analysis under 6%.

Structural characterization of natural human urinary and recombinant DNA-derived erythropoietin. Identification of des-arginine 166 erythropoietin.

Recombinant human erythropoietin (rhEPO) has been purified to apparent homogeneity from a Chinese hamster ovary cell line expressing a cDNA clone of the human gene. NH2-terminal sequencing of the recombinant hormone indicates that the 27-residue leader peptide is correctly and consistently cleaved during secretion of the recombinant protein into conditioned medium, yielding the mature NH2 terminus (Ala-Pro-Pro-Arg...). Analysis of the COOH terminus of rhEPO by peptide mapping and fast atom bombardment mass spectrometry (FABMS) demonstrates that the arginyl residue predicted to be at the COOH terminus (based on confirmation of both genomic and cDNA sequences) is completely missing from the purified protein. The truncated form of the recombinant hormone, designated des-Arg166 rhEPO, displays an in vivo specific activity of greater than 200,000 units/mg protein. Structural characterization of natural human urinary EPO (uEPO) by peptide mapping and FABMS reveals that the urinary hormone is also missing the COOH-terminal Arg166 amino acid residue, a modification that remained undetected until now. There is no evidence of further proteolytic processing at the COOH terminus beyond specific removal of the Arg166 amino acid residue in either rhEPO or uEPO. On the basis of the FABMS data, we propose that the physiologically active form of the hormone circulating in plasma and interacting with target cells in vivo is des-Arg166 EPO.

Analysis of the primary sequence of human myelin basic protein peptides 1-44 and 90-170 by fast atom bombardment mass spectrometry.

The originally described sequence of human myelin basic protein peptide 45-89 has recently been shown to contain two errors which have now been resolved. In the present study fast atom bombardment mass spectrometry was utilized to analyze the primary sequence of the other portions, peptides 1-44 and 90-170 of human myelin basic protein. The results obtained confirm the accuracy of the primary sequence published for both of these terminal peptides.

Structure-function relationships in human interleukin-11. Identification of regions involved in activity by chemical modification and site-directed mutagenesis.

Chemical modification approaches combined with site-directed and deletion mutagenesis have been used to identify functionally critical regions/residues of recombinant human IL-11 (rhIL-11). Incubation of rhIL-11 with iodoacetic acid results in specific alkylation of a single methionine residue, Met58, and a 25-fold reduction of in vitro biological activity on mouse plasmacytoma cells. A similar decrease in activity is observed when Met58 is substituted with Ala, Leu, Gln, Glu, or Lys by site-directed mutagenesis. Treatment of rhIL-11 with succinic anhydride leads to modification of the amino-terminal amino group and partial labeling of 2 lysines, Lys41 and Lys98, and to a 3-fold decrease in activity. The activity losses can be attributed to modification of the lysine residues, since the succinyl derivative of the amino terminus is fully active. In addition, carboxyl-terminal deletion mutagenesis studies have demonstrated that removal of the last 4 residues reduces rhIL-11 activity 25-fold, whereas removal of 8 or more amino acids results in an inactive molecule. Based on secondary structure predictions and the location of exon/intron boundaries in the IL-11 genomic structure, we propose a four-helix bundle topology as a structural model for rhIL-11. This model has been tested by limited proteolysis using three side chain-specific endoproteinases. A limited number of protease-sensitive cleavage sites are present in rhIL-11, and all but two are located in the postulated helix interconnecting loops or at helix termini. alpha-Helices, which in the proposed structure form a compact core of the molecule, are inaccessible to digestion under limiting conditions. According to the model, Met58, Lys41 and Lys98 are located on the surface of the molecule, in agreement with their preferential accessibility to chemical modifications. By analogy with human growth hormone, we postulate that Met58 and the carboxyl terminus of rhIL-11 are involved in the primary receptor binding site (site I), whereas Lys41 and Lys98 may be a part of binding site II.

The structure of tyrosine aminotransferase. Evidence for domains involved in catalysis and enzyme turnover.

The primary structure of tyrosine aminotransferase, as deduced from the nucleotide sequence of complementary DNA, was confirmed by fast atom bombardment mass spectrometry of tryptic peptides derived from the purified protein. Limited digestion of the native enzyme with trypsin released an acetylated, amino-terminal peptide; the new amino terminus in the modified enzyme was Val65. Endogenous proteases generated a chromatographically separable form of tyrosine aminotransferase that began at Lys35. Neither trypsin nor the other proteases altered the catalytic activity of tyrosine aminotransferase. Reduction of the holoenzyme with sodium borohydride yielded a major tryptic peptide containing phosphopyridoxamine bound to lysine 280, which probably functions in transamination. The carboxyl terminus of tyrosine aminotransferase contains features that typify proteins with short half-lives; it includes two negatively charged, hydrophilic segments that are enriched for glutamyl residues and are similar to a PEST region in ornithine decarboxylase (Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234, 364-368). Tyrosine aminotransferase belongs to a superfamily of enzymes which includes aspartate aminotransferase and can be aligned so that many invariant, functional residues coincide. Like the isoenzymes of aspartate aminotransferase, tyrosine aminotransferase may contain two domains, with a central, catalytic core, and a small domain made up of both amino- and carboxyl-terminal components. We speculate that the exposed small domain may confer the unusually rapid degradative rate that characterizes this enzyme.

Liquid chromatographic profile classification of acute and chronic leukemias.

Reversed-phase high performance liquid chromatography and multivariate discriminant analysis are used for the classification of acute and chronic leukemias. Plasma or serum profiles, mainly of nucleosides, bases, and aromatic amino acids, are segmented into specific retention time intervals. Peak areas in each retention time interval are summed such that the chromatographic profile can be represented as a pattern vector formed from the linear combination of peak areas. The preprocessing techniques of autoscaling and variance weighting minimized inadvertent weighting and reduced the contribution of nondescriptive data components in the development of the discriminant function. The acute lymphocytic leukemia plasma samples and controls were classified with 100% sensitivity and specificity. Chronic leukemia serum samples and controls were categorized with sensitivity and specificity values of 93.7 and 87.6%, respectively.

Rat hepatic cytosolic phosphoenolpyruvate carboxykinase (GTP). Structures of the protein, messenger RNA, and gene.

The primary structure of the messenger RNA coding for cytosolic phosphoenolpyruvate carboxykinase was determined by sequencing cDNA and genomic DNA and by primer extension of the mRNA. The molecule is 2624 nucleotides in length; this includes 143 nontranslated nucleotides at the 5' end and 615 nontranslated nucleotides at the 3' end. The 3' nontranslated sequence contains a 102-base pair region of alternating purine-pyrimidine nucleotides (the majority of which are UpG dinucleotides), several direct repeats and palindromic sequences, and 8 CpG dinucleotides. The corresponding segment of the phosphoenolpyruvate carboxykinase gene thus has characteristics which favor the formation of Z-DNA. The amino acid sequence of phosphoenolpyruvate carboxykinase was deduced from the mRNA sequence and confirmed by fast atom bombardment mass spectrometric analysis of peptides generated with trypsin and Staphylococcus aureus V8 protease. The protein consists of 621 amino acids and has a molecular weight of 69,289. Charon 4A lambda bacteriophage clones containing genomic DNA coding for phosphoenolpyruvate carboxykinase were isolated from a library of partial HaeIII digests of rat liver DNA. Two clones, lambda PC112 and lambda PC103, contained the entire coding region in 15-kilobase inserts and were used to subclone the gene into pBR322 as EcoRI, BamHI, or SstI-KpnI fragments. Using these subclones, the structure of the phosphoenolpyruvate carboxykinase gene was determined by S1 nuclease mapping, R-loop analysis, and DNA sequencing. The gene is composed of 10 exons and 9 introns with a total length of 6.0 kilobases. The transcription initiation site of the gene was determined by a combination of in vitro transcription in a HeLa cell lysate system, primer extension of mRNAPEPCK, and S1 nuclease mapping. In vitro transcription of purified DNA templates revealed three RNA polymerase II-dependent start sites. Two sites were separated by 600 base pairs on the coding strand and the third site was on the noncoding strand. The products of S1 nuclease mapping and primer extension from a BglII site were compared in order to determine which of the coding strand initiation sites was expressed in vivo. In both cases a 69-base pair fragment was generated and the 5' end of this corresponded to a thymidine residue identified in a sequence ladder of the genomic DNA coding strand. We conclude that mRNAPEPCK synthesis initiates with an adenine residue 69 base pairs 5' of the BglII site; this corresponds to the 3' most transcription initiation site determined in vitro.