Determination of partial amino acid composition from tandem mass spectra for use in peptide identification strategies

We demonstrate a new approach to the determination of amino acid composition from tandem mass spectrometrically fragmented peptides using both experimental and simulated data. The approach has been developed to be used as a search-space filter in a protein identification pipeline with the aim of increased performance above that which could be attained by using immonium ion information. Three automated methods have been developed and tested: one based upon a simple peak traversal, in which all intense ion peaks are treated as being either a b- or y-ion using a wide mass tolerance; a second which uses a much narrower tolerance and does not perform transformations of ion peaks to the complementary type; and the unique fragments method which allows for b- or y-ion type to be inferred and corroborated using a scan of the other ions present in each peptide spectrum. The combination of these methods is shown to provide a high-accuracy set of amino acid predictions using both experimental and simulated data sets. These high quality predictions, with an accuracy of over 85%, may be used to identify peptide fragments that are hard to identify using other methods. The data simulation algorithm is also shown post priori to be a good model of noiseless tandem mass spectrometric peptide data.     

Ca2+-surfactant interactions affect enzyme stability in detergent solutions

Detergent proteases and amylases generally bind Ca(2+) ions. These bound ions enhance enzyme stability, reducing the rates of degradative reactions such as unfolding and proteolysis. Thus, surfactant aggregates, such as micelles, affect protease and amylase stability indirectly, by competing with the enzymes for Ca(2+) ions. Dissociation constants for Ca(2+) interactions with anionic surfactant micelles are in the 10(-3) to 10(-2) M range. These interactions are weak relative to enzyme-Ca(2+) interactions (K(d) of order 10(-6) M). However, surfactant is typically present at much higher concentration than enzyme, and it is the Ca(2+)-micelle equilibrium that largely determines the amount of free Ca(2+) available for binding to enzymes. The problem of surfactant-mediated Ca(2+) removal from enzymes can be avoided by adding calcium to a detergent formulation in an amount such that the concentration of free Ca(2+) is around 10(-5)M.     

Dimeric galectin-1 binds with high affinity to alpha2,3-sialylated and non-sialylated terminal N-acetyllactosamine units on surface-bound extended glycans

Galectin-1 is a member of the galectin family of glycan-binding proteins and occurs as an approximately 29.5-kDa noncovalent homodimer (dGal-1) that is widely expressed in many tissues. Here, we report that human recombinant dGal-1 bound preferentially and with high affinity (apparent K(d) approximately 2-4 microM) to immobilized extended glycans containing terminal N-acetyllactosamine (LN; Galbeta1-4GlcNAc) sequences on poly-N-acetyllactosamine (PL; (-3Galbeta1-4GlcNAcbeta1-)(n)) sequences, complex-type biantennary N-glycans, or novel chitin-derived glycans modified to contain terminal LN. Although terminal Gal residues are important for dGal-1 recognition, dGal-1 bound similarly to alpha3-sialylated and alpha2-fucosylated terminal LN, but not to alpha6-sialylated and alpha3-fucosylated terminal LN. The binding specificity of human recombinant dGal-1 was similar to that observed with purified bovine heart-derived dGal-1. Unexpectedly, dGal-1 bound free ligands in solution with relatively low affinity and displayed no preference for extended glycans, indicating that dGal-1 preferentially recognizes extended glycans only when they are surface-bound, such as found on cell surfaces. Human dGal-1 also bound to both native and desialylated human promyelocytic HL-60 cells with similar affinity as observed for immobilized long chain PL. Binding to these cells was reduced upon treatment with endo-beta-galactosidase, which cleaves PL sequences, indicating that cell-surface PLs are ligands. To test the role of dimerization in dGal-1 binding, we examined the binding of a mutated form of dGal-1 that weakly dimerizes (monomeric Gal-1 (mGal-1)) and a covalently dimerized (chemically cross-linked) form of mGal-1 (cd-mGal-1). dGal-1 and cd-mGal-1 had similar affinities that were both approximately 3.5-fold higher for immobilized PL than observed for mGal-1, suggesting that dGal-1 acts as a dimer to cross-link terminal LN units on immobilized PL. These results indicate that dGal-1 functions as a dimer to recognize LN units on extended PLs on cell surfaces.     

Specific sequences in the N and C termini of apolipoprotein A-IV modulate its conformation and lipid association

Apolipoprotein (apoA-IV) is a 376-residue exchangeable apolipoprotein that may play a number of important roles in lipid metabolism, including chylomicron assembly, reverse cholesterol transport, and appetite regulation. In vivo, apoA-IV exists in both lipid-poor and lipid-associated forms, and the balance between these states may determine its function. We examined the structural elements that modulate apoA-IV lipid binding by producing a series of deletion mutants and determining their ability to interact with phospholipid liposomes. We found that the deletion of residues 333-343 strongly increased the lipid association rate versus native apoA-IV. Additional mutagenesis revealed that two phenylalanine residues at positions 334 and 335 mediated this lipid binding inhibitory effect. We also observed that residues 11-20 in the N terminus were required for the enhanced lipid affinity induced by deletion of the C-terminal sequence. We propose a structural model in which these sequences can modulate the conformation and lipid affinity of apoA-IV.     

Adsorption of poly(ethylene glycol)-modified ribonuclease A to a poly(lactide-co-glycolide) surface

Protein adsorption is a source of variability in the release profiles of therapeutic proteins from biodegradable microspheres. We employ optical reflectometry and total internal reflection fluorescence to explore the extent and kinetics of ribonuclease A (RNase A) adsorption to spin-cast films of poly(lactide-co-glycolide) (PLG) and, in particular, to determine how covalent grafting of polyethylene glycol (PEG) to RNase A affects adsorption. Adsorption kinetics on PLG surfaces are surface-limited for RNase A but transport-limited for unconjugated PEG homopolymers and for PEG-modified RNase A, indicating that PEG anchors the conjugates to the surface during the transport-limited regime. PEG modification of RNase A decreases the total number of adsorbed molecules per unit area but increases the areal surface coverage because the grafted PEG chains exclude additional surface area. Total internal reflection fluorescence-based exchange measurements show that there is no exchange between adsorbed and solution-phase protein molecules. This indicates an unusually tenacious adsorption. Streaming current measurements indicate that the zeta potential of the PLG surface becomes increasingly negative as the film is exposed to water for several weeks, as expected. Aging of the PLG surface results in increased adsorption of unmodified RNase A but decreased adsorption of unconjugated PEG homopolymers and of PEG-RNase A conjugates, relative to the extent of adsorption on freshly prepared PLG surfaces. Adsorption results correlate well with an increase in the rate, total extent and preservation of bioactivity of RNase A released from PLG microspheres for the PEG-modified version of RNase A.     

Laminin-sulfatide binding initiates basement membrane assembly and enables receptor signaling in Schwann cells and fibroblasts

Endoneurial laminins (Lms), beta1-integrins, and dystroglycan (DG) are important for Schwann cell (SC) ensheathment and myelination of axons. We now show that SC expression of galactosyl-sulfatide, a Lm-binding glycolipid, precedes that of Lms in developing nerves. This glycolipid anchors Lm-1 and -2 to SC surfaces by binding to their LG domains and enables basement membrane (BM) assembly. Revealingly, non-BM-forming fibroblasts become competent for BM assembly when sulfatides are intercalated into their cell surfaces. Assembly is characterized by coalescence of sulfatide, DG, and c-Src into a Lm-associated complex; by DG-dependent recruitment of utrophin and Src activation; and by integrin-dependent focal adhesion kinase phosphorylation. Collectively, our findings suggest that sulfated glycolipids are key Lm anchors that determine which cell surfaces can assemble Lms to initiate BM assembly and DG- and integrin-mediated signaling.     

The exocyst component Sec5 is present on endocytic vesicles in the oocyte of Drosophila melanogaster

The exocyst is an octameric complex required for polarized secretion. Some components of the exocyst are found on the plasma membrane, whereas others are recruited to Golgi membranes, suggesting that exocyst assembly tethers vesicles to their site of fusion. We have found that in Drosophila melanogaster oocytes the majority of the exocyst component Sec5 is unexpectedly present in clathrin-coated pits and vesicles at the plasma membrane. In oocytes, the major substrate for clathrin-dependent endocytosis is the vitellogenin receptor Yolkless. A truncation mutant of Sec5 (sec5(E13)) allows the formation of normally sized oocytes but with greatly reduced yolk uptake. We find that in sec5(E13) oocytes Yolkless accumulates aberrantly in late endocytic compartments, indicating a defect in the endocytic cycling of the receptor. An analogous truncation of the yeast SEC5 gene results in normal secretion but a temperature-sensitive defect in endocytic recycling. Thus, the exocyst may act in both Golgi to plasma membrane traffic and endocytic cycling, and hence in oocytes is recruited to clathrin-coated pits to facilitate the rapid recycling of Yolkless.     

Effects of the olfactory environment and nutrition on the ability of male Mediterranean fruit flies to endure starvation

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), is targeted for control using the sterile insect technique (SIT). For this technique to succeed, released males must be able to compete with wild males for copulations. Male success is mediated by survival in the field often in adverse conditions. Manipulation of the postteneral environment experienced by sterile males before release has been shown to affect male sexual success and survival. The objectives of this study were to determine how various diets, combined with exposure to volatiles containing alpha-copaene, affect the ability of male Mediterranean fruit flies (from a wild and two unisexual strains) to withstand starvation. Accordingly, we maintained males on one of eight regimes combing a diet of either sugar, sugar and protein, a protein pulse or apricot, with or without the aroma of the sexual stimulant alpha-copaene. The apricot diet was associated with the lowest ability to resist starvation. The sugar-only diet was associated with the highest ability to resist starvation by sterile males. Exposure to alpha-copaene, in combination with the apricot diet, had a significant negative effect on the ability of males (from all strains) to resist starvation relative to other regimes examined. We conclude that the holding regimes that elicit the best sexual performance from males paradoxically also hasten their demise, probably by initiating an irreversible metabolic cascade. The search for the optimal prerelease regime continues.