Characterization of disulfide bond position in proteins and sequence analysis of cystine-bridged peptides by tandem mass spectrometry.

Tandem mass spectrometry employing high-energy, collisionally activated dissociation (CAD) is shown to be a useful method for sequencing through the cystine bridge of intermolecularly disulfide-bonded peptides. A characteristic triplet of intense fragment ions is observed corresponding to cleavage through and to either side of the disulfide bridge. These fragments define the masses of the linked peptides. Fragments due to peptide chain cleavage are also observed at lower abundance in the product-ion spectra and can be sufficient to sequence both of the disulfide-linked peptides without any prior knowledge of the peptide or protein sequence. Even in cases where the peptide sequence-related product-ion yields are poor, the intensities of the disulfide cleavage ions are usually sufficient to determine the molecular weights of the component cystine-bridged peptides. In this paper we demonstrate that the high-energy CAD tandem MS approach may be used to characterize disulfide-bonded peptides directly in complex enzymatic or chemical digests of native proteins. This obviates the need for individual purification of intermolecularly disulfide-linked peptides prior to analysis. The techniques are illustrated here for synthetic, inter- and intramolecularly disulfide-linked peptides and for human transforming growth factor-alpha (des-Val-Val-TGF-alpha), a compact protein containing 48 residues and three disulfides.     

3D 13C/1H NMR-based assignments for side-chain resonances of Lactobacillus casei dihydrofolate reductase. Evidence for similarities between the solution and crystal structures of the enzyme

Summary ¹³C-based three-dimensional ¹H–¹H correlation experiments have been used to determine essentially complete ¹³C and ¹H resonance assignments for the amino acid side chains of uniformly ¹³C/¹⁵N labelled L. casei dihydrofolate reductase in a complex with the drug methotrexate. Excellent agreement is observed between these assignments and an earlier set of partial assignments made on the basis of correlating nuclear Overhauser effect and crystal structure data, indicating that the tertiary structure of the enzyme is similar in solution and in the crystal state.To whom correspondence should be addressed.     

Tissue-specific effects of allergic rhinitis in mouse nasal epithelia

Allergic rhinitis (AR) can cause significant olfactory loss, but few studies have specifically investigated AR effects on olfactory and nasal respiratory tissues per se. To address this, we used a murine AR protocol employing nasal allergen infusion for both sensitization and challenges. Seven- to 11-week BALB/c mice were bilaterally infused with 1% ovalbumin (OVA) in phosphate-buffered saline (PBS) or PBS alone for 6 or 11 weeks, given single bilateral PBS or OVA infusions 24 h before sacrifice, or left untreated. High OVA-specific IgE serum levels and eosinophil infiltration confirmed AR induction. Olfactory (OE) and respiratory (RE) epithelia showed distinctly different responses, most conspicuously, massive eosinophil infiltration of immediately RE-subjacent lamina propria. In OE, such infiltration was minimal. Significant RE hypertrophy and hyperplasia also occurred, although OE organization was generally maintained and extensive disruption localized despite a 20% reduction in sensory neurons and globose basal cells after 11 weeks OVA. Pronounced Bowman's gland hypertrophy crowded both OE and olfactory nerve bundles. Cellular proliferation was widely distributed in RE but in OE was localized to normally thinner OE and RE-proximal OE, suggesting possible indirect RE influences. Terminal deoxynucleotide transferase (TdT) nick end labeling was greater in OE than RE and, in contrast to other effects, occurred with acute infusions and chronic PBS alone, often unilaterally. Following chronic OVA, AR-related bilateral increases appeared superimposed on those. These findings indicate AR effects on olfactory function may be complex, reflecting various levels of RE/OE responses and interactions.     

Deep,Quantitative Coverage of the Lysine Acetylome Using Novel Anti-acetyl-lysine Antibodies and an Optimized Proteomic Workflow

Introduction of antibodies specific for acetylated lysine has significantly improved the detection of endogenous acetylation sites by mass spectrometry. Here, we describe a new, commercially available mixture of anti-lysine acetylation (Kac) antibodies and show its utility for in-depth profiling of the acetylome. Specifically, seven complementary monoclones with high specificity for Kac were combined into a final anti-Kac reagent which results in at least a twofold increase in identification of Kac peptides over a commonly used Kac antibody. We outline optimal antibody usage conditions, effective offline basic reversed phase separation, and use of state-of-the-art LC-MS technology for achieving unprecedented coverage of the acetylome. The methods were applied to quantify acetylation sites in suberoylanilide hydroxamic acid-treated Jurkat cells. Over 10,000 Kac peptides from over 3000 Kac proteins were quantified from a single stable isotope labeling by amino acids in cell culture labeled sample using 7.5 mg of peptide input per state. This constitutes the deepest coverage of acetylation sites in quantitative experiments obtained to-date. The approach was also applied to breast tumor xenograft samples using isobaric mass tag labeling of peptides (iTRAQ4, TMT6 and TMT10-plex reagents) for quantification. Greater than 6700 Kac peptides from over 2300 Kac proteins were quantified using 1 mg of tumor protein per iTRAQ 4-plex channel. The novel reagents and methods we describe here enable quantitative, global acetylome analyses with depth and sensitivity approaching that obtained for other well-studied post-translational modifications such as phosphorylation and ubiquitylation, and should have widespread application in biological and clinical studies employing mass spectrometry-based proteomics.Lysine acetylation (Kac)¹ is a well conserved, reversible post-translational modification (PTM) involved in multiple cellular processes (1). Acetylation is regulated by two classes of enzymes: lysine acetyltransferases (KATs) and histone deacetylases (HDACs) (2–4). This modification was originally identified as a nuclear event on histone proteins and has been long appreciated for its role in epigenetic and DNA-dependent processes. With the help of a growing number of large-scale acetylation studies, it has become evident that lysine acetylation is ubiquitous, also occurring on cytoplasmic and mitochondrial proteins and has a role in signaling, metabolism, and immunity (1, 4–6). Therefore, the examination of lysine acetylation on nonhistone proteins has gained a prominent role in PTM analysis.To date, the identification of large numbers of acetylation sites has been challenging because of the substoichiometric nature of this modification (7, 8). Additionally, global acetylation is generally less abundant than phosphorylation and ubiquitylation (1). The introduction of antibodies specific for lysine acetylation has significantly improved the ability to enrich and identify thousands of sites (9–14). A landmark study by Choudhary et al. used anti-Kac antibodies to globally map 3600 lysine acetylation sites on 1750 proteins, thereby demonstrating the feasibility of profiling the acetylome (10). A more recent study by Lundby et al. investigated the function and distribution of acetylation sites in 16 different rat tissues, and identified, in aggregate, 15,474 acetylation sites from 4541 proteins (12).Although anti-acetyl lysine antibodies have been a breakthrough for globally mapping acetylation sites (9–12), it remains a challenge to identify large numbers of lysine acetylation sites from a single sample, as is now routinely possible for phosphorylation and ubiquitylation (13, 15–18). To improve the depth-of-coverage in acetylation profiling experiments there is a clear need for (1) alternative anti-acetyl lysine antibodies with higher specificity, (2) optimized antibody usage parameters, and (3) robust proteomic workflows that permit low to moderate protein input. In this study, we describe a newly commercialized mixture of anti-Kac antibodies and detail a complete proteomic workflow for achieving unprecedented coverage of the acetylome from a single stable isotope labeling by amino acids in cell culture (SILAC) labeled sample as well as isobaric tags for relative and absolute quantitation (iTRAQ)- and tandem mass tag (TMT)-labeled samples.     

Different NK cell surface phenotypes defined by the DX9 antibody are due to KIR3DL1 gene polymorphism

KIR3DL1 and KIR3DL2 are NK cell receptors for polymorphic HLA-B and -A determinants. The proportion of NK cells that bind anti-KIR3DL1-specific Ab DX9 and their level of binding vary between individuals. To determine whether these differences are due to KIR polymorphism, we assessed KIR3D gene diversity in unrelated individuals and families. Both KIR3DL1 and KIR3DL2 are highly polymorphic genes, with KIR3DS1 segregating like an allele of KIR3DL1. A KIR haplotype lacking KIR3DL1 and KIR3DS1 was defined. The two KIR3DL1 alleles of a heterozygous donor were expressed by different, but overlapping, subsets of NK cell clones. Sequence variation in KIR3DL1 and KIR3DL2 appear distinct; recombination is more evident in KIR3DL1, and point mutation is more evident in KIR3DL2. The KIR3DL1 genotype correlates well with levels of DX9 binding by NK cells, but not with the frequency of DX9-binding cells. Different KIR3DL1 alleles determine high, low, and no binding of DX9 Ab. Consequently, heterozygotes for high and low binding KIR3DL1 alleles have distinct subpopulations of NK cells that bind DX9 at high and low levels, giving characteristic bimodal distributions in flow cytometry. The Z27 Ab gave binding patterns similar to those of DX9. Four KIR3DL1 alleles producing high DX9 binding phenotypes were distinguished from four alleles producing low or no binding phenotypes by substitution at one or more of four positions in the encoded protein: 182 and 283 in the extracellular Ig-like domains, 320 in the transmembrane region, and 373 in the cytoplasmic tail.     

Changes in TGF-β receptors of rat hepatocytes during primary culture and liver regeneration: Increased expression of TGF-β receptors associated with increased sensitivity to TGF-β-mediated growth inhibition

To clarify the role of transforming growth factor-β (TGF-β) and its receptors in hepatocyte growth, we studied the expression of TGF-β1 and its receptors and the sensitivity to growth inhibition by TGF-β1 protein in rat hepatocytes derived from resting and regenerating livers. In hepatocytes derived from resting livers, mRNAs for TGF-β type II receptor (TβR-II), insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M-6-PR), and TGF-β1 increased with time in primary culture. The cell surface TGF-β receptor proteins (TβR-I, II, and III), examined by the receptor affinity-labeling assay using ¹²⁵I-TGF-β1, also increased, especially after 48 hr of culture. Hepatocytes were more sensitive to inhibition of DNA synthesis, when the TGF-β1 protein was added at later times in culture, corresponding to the presence of increased TGF-β receptors. In hepatocytes from regenerating livers after a partial hepatectomy (PH), an increase of TβR-I, TβR-II, TβR-III, IGF-II/M-6-PR, and TGF-β1 mRNAs was found, compared with hepatocytes from resting livers. Similarly, using TGF-β receptor affinity-labeling assay, hepatocytes from PH livers were found to have an increase in TβR-I, II, and III proteins, with a peak at 4 days post-PH, compared with hepatocytes from resting livers. When TGF-β1 protein was added for a short period (6 or 24 hr) after cell attachment to hepatocyte cultures, it inhibited DNA synthesis more effectively in hepatocytes from regenerating compared with resting livers. Our results show that hepatocyte TGF-β receptors and sensitivity to growth inhibition by TGF-β1 protein change together and are modulated during liver regeneration, as well as during the conditions of primary culture. J. Cell. Physiol. 176:612–623, 1998. © 1998 Wiley-Liss, Inc.     

Effect of canavanine and other amino acid analogues on akinete formation in the cyanobacterium Anabaena cylindrica

Addition of the arginine analogue, canavanine, to cultures of nitrogen-fixing Anabaena cylindrica at the onset of akinete formation, resulted in the development of akinetes randomly distributed within the filament, in addition to those adjacent to heterocysts. The total frequency of akinetes increased up to five-fold. A feature of akinetes is their increased content of cyanophycin granules (an arginine-aspartic acid polymer) and addition of canavanine to cultures at an earlier stage resulted in entire filaments becoming agranular and containing agranular akinetes. The effects on akinete pattern appeared to be specific for canavanine since other amino acid analogues, although increasing the frequency of akinetes (approximately two-fold), had no effect on their position relative to heterocysts. In ammonia-grown, stationary phase cultures of A. cylindrica, akinetes were observed adjacent to proheterocysts and in positions more than 20 cells from any heterocyst. These observations indicate that nitrogen fixation and heterocysts are not essential for akinete formation in A. cylindrica, although the availability of a source of fixed nitrogen does appear to be a requirement.These results suggest that during exponential growth some aspect of the physiology of vegetative cells suppresses their development into akinetes and that the role of the heterocyst may not be one of direct stimulation of adjacent vegetative cells to form akinetes, but the removal or negation of the inhibition within them. A model for akinete formation and the involvement of canavanine is given.     

Unforeseen consequences of IL-12 expression in the eye of GFAP-IL12 transgenic mice following herpes simplex virus type 1 infection

Transgenic mice expressing interleukin-12 (IL-12) under the glial fibrillary acidic protein (GFAP) promoter were evaluated for their sensitivity to herpes simplex virus type 1 (HSV-1) infection of the cornea. There was a modest but significant decrease in the infiltration of mononuclear cells in the cornea of the GFAP-IL12 transgenic mice compared to the wild-type controls during the acute stage of infection. However, during the latent stage of infection (i.e., day 30 postinfection) GFAP-IL12 transgenic mice had significantly more infiltrating cells in the corneal stroma compared to the wild-type controls. The infiltration was exacerbated by depleting transgenic mice of either CD4(+) or CD8(+) cells at the time of infection. In addition, infiltration of mononuclear cells was associated with the expression of transforming growth factor-beta (TGF-beta) by cells in the cornea. Consistent with increases in tissue associated TGF-beta was the presence of anterior subcapsular cataracts in the GFAP-IL12 transgenic mice. Although the GFAP-IL12 transgenic mice are highly resistant to HSV-1 infection in the eye, this resistance is not related to local expression of TGF-beta1 per se because transgenic mice expressing TGF-beta1 driven by the lens-specific alphaA-crystallin promoter succumb to HSV-1 infection at a similar rate as wild-type controls.     

Rapid method for measuring ScFv thermal stability by yeast surface display

We have characterized a simplified method to determine the relative thermal stability of single-chain antibodies by following the irreversible denaturation of scFv fusions on the surface of yeast by flow cytometry. The method was highly reproducible and correlated well with other methods used to monitor thermal denaturation of the soluble proteins. We found a range of thermal stabilities for wild-type single-chain antibodies with half-maximum denaturation temperatures between 43 and 61 degrees C. The ability to quantitate thermal stability of antibodies or other proteins that are immobilized on the surface of yeast allows rapid comparisons of primary structural information with stability. Thermal denaturation could be a useful parameter to consider in the choice of scFv fragments for various applications.