Kristine M. Swiderek

Gallinacins : cysteine-rich antimicrobial peptides of chicken leukocytes

We purified three homologous antimicrobial peptides (‘gallinacins’) from chicken leukocytes, examined their antimicrobial activity in vitro, and established their primary sequences by a combination of gas phase microsequencing and on‐line LC‐ESI‐MS analysis of endo‐ and exoprotease peptide digests. The peptides contained 36–39 amino acid residues, were relatively cationic due to their numerous lysine and arginine residues, and each contained 3 intramolecular cystine disulfide bonds. Gallinacins showed primary sequence homology to the recently delineated β‐defensin family, heretofore found only in the respiratory epithelial cells and neutrophils of cattle, suggesting that β‐defensins originated at least 250 million years ago, before avian and mammalian lineages diverged. The 9 invariant residues (6 cysteines, 2 glycines and 1 proline) common to avian gallinacins and bovine β‐defensins are likely to constitute the essential primary structural motif of this ancient family of host‐defense peptides.

Amino acid sequence of CAP2b, an insect cardioacceleratory peptide from the tobacco hawkmoth Manduca sexta

The primary structure of a novel insect neuropeptide, Cardioacceleratory Peptide 2b (CAP2b), from the tobacco hawkmoth Manduca sexta has been established using a combination of mass spectroscopy, Edman degradation microsequencing, amino acid analysis, and biological assays. The sequence of CAP2b, pyroGlu‐Leu‐Tyr‐Ala‐Phe‐Pro‐Arg‐Val‐amide, has a molecular weight of 974.6 and is blocked at both the amino and car☐yl ends. Examination of several national computer protein data bases failed to reveal other peptides or proteins with any sequence homology to CAP2b indicating that this is likely to be a novel insect neuropeptide. This peptide may be a general activator of insect viscera since it causes an increase in heart rate in Manduca and in Drosophila, and has also been implicated in the regulation of fluid secretion by the Malphigian tubules of Drosophila.

Prophenin-1, an exceptionally proline-rich antimicrobial peptide from porcine leukocytes

We purified and characterized an unusual antimicrobial peptide, prophenin‐1 (PF‐1), from porcine leukocytes. The peptide had a mass of 8,683 and contained 79 residues, including 42 (53.2%) prolines and 15 (19.0%) phenylalanines. Its N‐terminal 60 residues consisted of three perfect and three nearly perfect repeats of a decamer, FPPPNFPGPR. Prophenin‐1 was encoded on a cathelin‐containing precursor and showed substantially more activity against E. coli, a Gram‐negative bacterium, than against Listeria monocytogenes, a Gram‐positive organism, in vitro.

Selenium-dependent glutathione peroxidase-GI is a major glutathione peroxidase activity in the mucosal epithelium of rodent intestine

Gpx2 mRNA, encoding a selenium-dependent glutathione peroxidase (GPX-GI), has been found to be highly expressed in the gastrointestinal tract (GI) mucosal epithelium. In this study, we show that GPX-GI is produced in the mucosal epithelium of the adult rat GI tract and that the activity levels are comparable to that from GPX-1. Post-mitochondrial supernatant GPX activity from the mucosal epithelium of the complete length of the small intestine was partially purified. A sample enriched for putative GPX-GI was fractionated by SDS-polyacrylamide gel electrophoresis. Polypeptides of 21 kDa and 22 kDa were digested with trypsin. After resolving the tryptic peptides by high pressure liquid chromatography (HPLC), the major peaks were analyzed for their amino acid sequence by Microflow-HPLC-Tandem Mass Spectrometry and automated Edman degradation sequencing. Both methods revealed that the 21-kDa sample contained rat GPX-GI determined by the sequence homology with the deduced mouse GPX-GI polypeptide sequence. Rat GPX-1 was also detected in the samples. AntiGPX-GI and antiGPX-1 antibodies were used to determine the distribution of the respective isoenzyme activities along the length of the intestine and with respect to the crypt to villus axis in rats. GPX-GI and GPX-1 activities were uniformly distributed in the middle and lower GI tract and with respect to the crypt to villus axis. GPX-GI activity accounted nearly the same percentage of the total GPX activity as GPX-1 in all of the these compartments. Studies on the distal ileum segment of wildtype and Gpx1 gene knockout mice showed that GPX-GI activity was also at parity with GPX-1 in the mucosal epithelium of this segment.

Structure-function studies of human aromatase ☆

Site-directed mutagenesis experiments have been carried out to determine the structure-function relationship of human aromatase. By sequence comparison, the region in aromatase that corresponds to the distal helix of cytochrome P-450cam has been identified to be Gln-298 to Val-313. Eight aromatase mutants with changes in this region, i.e. C299A, E302L, P308F, D309N, D309A, T310S, T310C, and S312C, have been generated using a mammalian cell stable-expression system. The results from site-directed mutagenesis studies indicate that the region containing Gln-298 to Val-313 is indeed a very important part of the active site of aromatase. The catalytic properties of P308F, D309N, and D309A have been examined in detail and are discussed. Active site-directed labeling is also an important approach to investigate the structure-function relationship of aromatase. HPLC-linked electrospray mass spectrometry is indicated as a useful technique for the characterization of active site-directed probe-modified enzyme. The mass spectral analysis of aromatase suggests that aromatase is glycosylated.

Trace structural analysis of proteins.

Publisher Summary With the introduction of mass spectrometry (MS) into protein chemistry, a new, powerful tool has been implemented to perform different structural analyses on small amounts of samples. The analysis of macromolecules, with electrospray mass spectrometry, in combination with liquid chromatography, within an error of ±0.01%, is a reliable technique for mass determination. Accurate mass determination of a protein, with known primary structure, can indicate possible posttranslational modifications of the protein as well as errors in protein sequence analysis or complementary DNA (cDNA)-derived sequences. The success of a variety of experiments, such as site-directed mutagenesis or covalent modification studies, can be verified quickly without employing large amounts of sample. This chapter explains examples for a variety of structural analyses of low amounts of proteins and peptides, utilizing different mass spectrometric techniques, in combination with micro-HPLC (high-performance liquid chromatography). For demonstration of the different techniques standard proteins as well as “real-life” samples are used. The standard protein used is horse cytochrome c, digested with the endoproteinase Lys-C. This chapter discusses the possible strategies to solve some often encountered structural problems.

Specific binding of glucosaminylmuramyl peptides to histones

Intracellular N‐acetylglucosaminylmuramyl peptide‐binding proteins of murine macrophages and myelomonocytic WEHI‐3 cells were characterized. SDS‐PAGE and Western blotting revealed proteins with molecular masses of 18, 32 and 34 kDa retaining the ability to specifically bind glucosaminylmuramyl dipeptide. The inhibition analysis demonstrated that only biologically active muramyl peptides but not inactive analogs or fragments of glucosaminylmuramyl dipeptide could inhibit glucosaminylmuramyl dipeptide‐binding to these proteins. Purification of these proteins and sequencing of peptides obtained after in‐gel trypsin digestion enabled us to identify the above mentioned proteins as histones H1 and H3. These findings suggest that nuclear histones might be target molecules for muramyl peptides.

Strategies for the removal of ionic and non-ionic detergents from protein and peptide mixtures for on- and off-line liquid chromatography mass spectrometry (LCMS)

Publisher Summary Ionic and non-ionic detergents are reagents widely used during the purification of proteins and peptides; the presence of ionic detergents, such as SDS is well known to destroy any chromatographic resolution on reversed phase HPLC even in very low concentrations, while the presence of many non-ionic detergents interferes with mass spectral analysis. This chapter describes some strategies to remove ionic and non-ionic detergents during sample preparation at the low picomole level for micro HPLC chromatography and mass spectrometry. SDS can be removed by precipitation with guanidine hydrochloride; protein can be precipitated with trichloroacetic acid (TCA), leaving for example SDS in the supernatant; however, these techniques bear die risk of losing the sample, especially if only picomoles of protein or peptide are available. LC-MS analysis of the separated peptides indicates that the chromatography of the sample with SDS is almost identical to the peptide separation without SDS. Applying this technique to samples contaminated with SDS will improve chromatography as well as mass spectral analysis. However, it is not to exclude that some peptides or proteins might interact with the pre-column and will be retained in the same manner as SDS.

Sera from children with type 1 diabetes mellitus react against a new group of antigens composed of lysophospholipids.

Several autoantibodies related to Type 1 diabetes mellitus and their corresponding autoantigens have been previously identified. While peptide antigens are more widely recognized, lipid antigens like sulfatides and gangliosides are also known epitopes for the diabetic humoral immune response. Islet cell antibodies (ICA) in Type 1 diabetes are heterogeneous immunoglobulins directed against selected antigens in the islets of Langerhans. Moreover, ICA may be the best predictive marker of disease in family members of patients with Type 1 diabetes. The aims of this study were: (1) to purify lipids from porcine pancreas that contain ICA epitopes; (2) to characterize these lipid antigens, and (3) to use the purified lipids in an assay to detect antibodies in patients with Type 1 diabetes. A unique family of 4 lysophospholipids, 1 fully characterized as lysophosphatidylmyoinositol, partially inhibited ICA staining, and therefore, were considered to be candidate antigens for an ICA immunoassay. Using a dot blot immunoassay, we detected antibodies directed against these phospholipids in 28 out of 46 (61%) diabetic sera, while detecting only 1 false positive out of 28 nondiabetic sera (3.6%; p < 0.0001 comparing diabetic vs. nondiabetic serum). Therefore, lysophospholipid immunoassay positivity is present in sera of Type 1 diabetic patients. Furthermore, we detected 15 out of 23 ICA-negative diabetic sera (65.2%), showing that our phospholipid immunoassay does not correlate with ICA positivity.

Comparison of ESI-MS, LSIMS and MALDI-TOF-MS for the primary structure analysis of a monoclonal antibody

Publisher Summary This chapter describes and compares the three most widely used techniques for the analysis of the heavy and light chains of the anti-CEA (carcinoembryonic antigen) antibody CEA11 H5; LSIMS, MALDI-TOF-MS, and ESI-MS. In LSIMS and MALDI, individual peptide fractions are analyzed by mixing with a matrix followed by ionization and mass analysis, while in ESI the samples can be separated on-line by LC, eliminating the need for individual peak collection. Of the three methods, ESI and MALDI have no problems with the analysis of very large peptides; however, this is an obvious limitation of LSIMS. MALDI is the simplest of the techniques, but required careful attention to calibration, usually requiring internal calibrants to obtain accurate masses. The effect of detergent on LSIMS and ESI is severe. MALDI is clearly the method of choice for samples, including detergents, but otherwise detergents should be avoided. The chapter concludes that mass analysis of peptides blotted and digested from SDS gels is a powerful, general approach, but requires much time and effort in sorting out the data no matter which mass spectrometric approach is used.