Barbara M. Merrill

Site-specific Conjugation on Serine → Cysteine Variant Monoclonal Antibodies

We have engineered a cysteine residue at position 442 (EU/OU numbering) in the third constant domain (CH3) of the heavy chain of several IgGs with different specificities, isoforms, and variants with the intent to introduce a site for chemical conjugation. The variants were expressed in NS0 mouse myeloma cells, where monomeric IgG is the major form and formation of aggregate was minimal. Monomeric IgG contained no free thiol; however, it was discovered that the engineered thiols were reversibly blocked and could be reduced under controlled conditions. Following reduction, reactive thiol was conjugated with a cysteine-specific bifunctional chelator, bromoacetyl-TMT to a humanized 323/A3 IgG4 variant. Conjugation had no significant effect on antibody affinity. To prove that the conjugation was site-specific, an antibody-TMT conjugate was labeled with lutetium-177 and subjected to peptide mapping followed by sequence analysis. Glu-C digestion demonstrated that 91% of the label was recovered in the COOH-terminal peptide fragment containing the engineered cysteine.

Mammalian single-stranded DNA binding protein UP I is derived from the hnRNP core protein A1.

Antibodies induced against mammalian single‐stranded DNA binding protein (ssDBP) UP I were shown to be cross‐reactive with most of the basic hnRNP core proteins, the main constituents of 40S hnRNP particles. This suggested a structural relationship between both groups of proteins. Using the anti‐ssDBP antibodies, a cDNA clone (pRP10) was isolated from a human liver cDNA library in plasmid expression vector pEX1. By DNA sequencing this clone was shown to encode in its 949 bp insert the last 72 carboxy terminal amino acids of the ssDBP UP I. Thereafter, an open reading frame continued for another 124 amino acids followed by a UAA (ochre) stop codon. Direct amino acid sequencing of a V8 protease peptide from hnRNP core protein A1 showed that this peptide contained at its amino terminus the last 11 amino acids of UP I followed by 19 amino acids which are encoded by the open reading frame of cDNA clone pRP10 immediately following the UP I sequence. This proves that ssDBP UP I arises by proteolysis from hnRNP core protein A1. This finding must lead to a re‐evaluation of the possible physiological role of UP I and related ssDBPs. The formerly assumed function in DNA replication, although not completely ruled out, should be reconsidered in the light of a possible alternative or complementary function in hnRNA processing where UP I could either be a simple degradation product of core protein A1 (as a consequence of controlling the levels of active A1) or may continue to function as an RNA binding protein which has lost the ability to interact with the other core proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Assignment of cysteine and tryptophan residues during protein sequencing: Results of ABRF-94SEQ

Publisher Summary Routine assignment of cysteine and tryptophan residues is necessary for completeness of protein sequencing, because cysteine residue provides the major covalent cross-linkage in proteins and contributes to the biological and catalytic activity of many folded polypeptides. While, accurate assignment of tryptophan residues is crucial, when sequencing is used to design oligonucleotide probes of limited redundancy. This chapter discusses methods to improve the reliability of cysteine and tryptophan assignment, and describes the results of ABRF-94SEQ. ABRF-94SEQ allows to see which methods are used successfully in other facilities, and also provide the opportunity to assess these methods. It is observed that some who alkylate and some who does not alkylate, take risks making positive assignments for cysteine, when no PTH-residue or dehydroalanine/DTT adduct is apparent. If this assumption is done, it must be noted that this is not a positive assignment. Although cysteine and tryptophan identification may be problematic, the data suggest that routine identification of these residues should be possible for the majority of facilities.

Evaluation of ABRF-96SEQ: A sequence assignment exercise

Publisher Summary The ability of most core laboratories to interpret data correctly from a homogenous and relatively high-level sample that has no amino acids that are difficult to identify, is of a finite length, and has clear PTH chromatograms is excellent. However, even under ideal conditions there usually remain a few positive incorrect calls that should have been reported as tentative. The overall accuracy decreases when the sample is low-level and complex. Association of Biomolecular Resource Facilities (ABRF)-96SEQ was designed to try to ascertain the source of these problems and to provide individual laboratories with a means of self-evaluation. ABRF-96SEQ offered a unique opportunity for participating laboratories ability to interpret the same data. Generally, the committee feels that there are more positive incorrect calls than there should have been, and urges all respondents that perform sequence analysis to be cautious in assigning positive correct calls, particularly at the end of the sequence. A comparative summary of the current study with previous ABRF-SEQ samples is shown in this chapter. It is evident that ABRF-96SEQA has virtually a 100% accuracy of positive calls compared with other studies indicating that a straightforward homogenous sample with no difficult amino acids, clear chromatography, and a finite ending poses no problems for data interpretation.

Design and analysis of ABRF-95SEQ, a recombinant protein with sequence heterogeneity

Publisher Summary This chapter summarizes the sequencing results for the current test sample, ABRF-95SEQ that represents the eighth in a series of unknown samples that have been distributed to Amino Acid Analysis Research Committee of the Association of Biomolecular Resource Facilities (ABRF) facilities that perform protein sequencing. The chapter assesses three important aspects of protein sequence analysis that would routinely be encountered in a typical core facility: (1) the length of sequence assignment possible with a low-level sample, (2) the reliability of cysteine and tryptophan identification, and (3) the ability of the members to recognize sample microheterogeneity. Results from ABRF-94SEQ and ABRF-95SEQ suggest that the accuracy of Trp assignments may depend in part on the position of the residue in a sequence, with earlier assignments being more accurate than later ones. Even though there are a number of simple procedures for modifying Cys, most facilities do not routinely use them, resulting in many inaccurate assignments for this residue. Sequence heterogeneity also reduces assignment accuracy but to a lesser extent. Thus, multiple factors play a role in the accuracy of sequence assignments.

Identification of a 7B2-derived tridecapeptide from bovine adrenal medulla chromaffin vesicles

Summary1.A novel tridecapeptide was isolated from extracts of bovine adrenal medulla chromaffin vesicles and the primary structure determined to be SVPHFSDEDKDPE.2.This peptide is identical to the C termini of human and porcine 7B2 and is highly homologous to the same region of the mouse andXenopus lavis protein.3.In all these species the homologous peptide is preceded by a pair of lysine residues, a potential proteolytic processing site.4.Ser6 is part of a well-conserved casein kinase II consensus phosphorylation sequence. Evidence for phosphorylation of this residue was obtained during Edman sequencing.5.Thus, this novel adrenal medullary probably arises from the posttranslational processing of the bovine 7B2 protein.

A novel tetradecapeptide isolated from bovine adrenal medulla chromaffin vesicles with strong homology to an internal sequence coded by the rat 1B1075 (Preprosecretogranin III) gene.

A novel tetradecapeptide, PheProLysProAlaGlySerGlnAspLysProLeuHisAsn, was isolated from boiling water extracts of bovine adrenal medulla chromaffin vesicles. The primary structure of the peptide was characterized by amino acid analysis, fast atom bombardment mass spectrometry, and gas-phase sequencing. The synthetic and native peptides comigrated on reversed-phase high-performance liquid chromatography, supporting the proposed sequence. This peptide shares 86% homology to residues 67-80 of the recently reported rat 1B1075 gene product secretogranin III and probably represents a processed product derived from the bovine equivalent.

Initial characterization of multiple endopeptidases in bovine adrenal chromaffin vesicles

All regulatory peptides are synthesized as large, inactive precursor proproteins that must undergo specific endoproteolytic processing to yield bioactive peptides. In most cases, enzymatic release of the biologically active peptides occurs by endoproteolytic cleavage at doublets of basic amino acid residues that precede and/or follow that particular sequence [1,2]. The catecholamine-containing chromaffin vesicles of the adrenal medulla are enriched in a great variety of regulatory peptides (i.e. enkephalins, neurotensin, neuropeptide Y, etc.) and thus are good sources for the isolation and characterization of peptide processing enzymes [3,4,5]. To isolate putative endopeptidases of the thioland serineprotease families, the dialysed lysate of purified bovine chromaffin vesicles was consecutively fractionated through p-chloro-mercuribenzoateagarose (PCMB-agarose), p-aminobenzamidineagarose (p-ABZ-agarose) and soybean trypsin inhibitor-agarose (STI-agarose) affinity columns. Three intermediate proenkephalin precursor peptides (BAM12P, BAM22P and amidorphin) were used as substrates for the assay of endopeptidase activities. These peptides contain pairs of basic amino acids, Arg-Arg, Lys-Arg and Lys-Lys, which have putative cleavage sites for the endopeptidases. Degradation peptide fragments were separated by reverse phase HPLC and identified by FAB mass spectrometry, amino acid analysis and sequence analysis, which was performed with an Applied Biosynthesis model 477a protein sequencer using Edman degradation chemistry. The fraction retained and eluted from PCMB-agarose affinity chromatography hydrolyzed the Arg-Arg sequence of BAM12P, resulting in the generation of Met-enkephalin and Met-enkephalin-Arg at pH 5.7. However, this enzyme preparation was unable to hydrolyze amidorphin at the Lys-Lys pair of basic residues. This activity was inhibited by PCMB and E64, indicating that a thiol protease is involved. The dialysate fraction that was not retained by the PCMB-agarose column was subsequently retained and eluted from the p-ABZagarose affinity column. This dialysate fraction contained enzyme activity which cleaved at the Lys-Arg of BAM22P and at the Lys-Lys of amidorphin at pH 7.4. BAM12P was however a poor substrate for this fraction. This activity was not inhibited by ST1, which is indicative of a non-trypsin-like endopeptidase. Additionally, a separate endopeptidase cleaving at Glu-Trp of BAM22P, resulting in the generation of BAM12P, was also found in this preparation. The dialysate fraction not retained in the first two columns but retained and eluted from the STI-agarose affinity column had an enzyme activity capable of hydrolyzing amidorphin at the carboxy side of Lys-Lys. This activity was completely inhibited by STI which is indicative of a trypsin-like endopeptidase. BAM12P however was poorly cleaved by this preparation. This study demonstrates that a variety of different endopeptidase activities is found in soluble lysates of adrenal medulla chromaffin vesicles. A multiplicity of peptide processing enzymes with different specificities suggests the possibility that modification of a particular processing enzyme may result in specific changes in the cocktail of regulatory peptides.

Use of HPLC Comparative Peptide Mapping in Structure/Function Studies

Among the numerous approaches that are frequently used to correlate a protein’s structure with its function are limited proteolysis to generate “active” fragments; chemical modification with group specific reagents, in vitro site directed mutagenesis and finally, covalent crosslinking of proteins to ligands so that individual amino acids near the active site of the protein can be identified. The ability of peptide mapping by reverse-phase HPLC to rapidly identify subtle alterations in the primary structures of proteins enables this technique to play a key role in these structure/function studies. In this report practical suggestions will lie given for obtaining complete enzymatic digests of proteins, eliminating HPLC baseline artifacts, maximizing the reproducibility of the resulting retention times, and gas phase sequencing of the HPLC purified peptides. These methods are generally applicable and should be of assistance in quickly bringing the full potential of HPLC comparative peptide mapping to bear on other questions concerning the interrelationship of protein structure and function. Specific applications will be given where these techniques have been used to rapidly verify the; location of amino acid substitutions resulting from in vitro mutagenesis, to identify an active site peptide in a ssDNA binding protein, and to demonstrate that the most abundant ssDNA binding protein in higher eucaryotes is actually a proteolytic fragment derived from a heterogeneous nuclear ribonucleoprotein (hnRNP).