Screening for N-glycosylated proteins by liquid chromatography mass spectrometry

In the last few years mass spectrometry has become the method of choice for characterization of post-translationally modified proteins. Whereas most protein chemical modifications are binary in the sense that only one change can be associated with a given residue, many different oligosaccharides can be attached to a glycosylation site residue. The detailed characterization of glycoproteins in complex biological samples is extremely challenging. However, information on N-glycosylation can be gained at an intermediary level. Here we demonstrate a procedure for mapping N-glycosylation sites in complex mixtures by reducing sample complexity and enriching glycoprotein content. Glycosylated proteins are selected by an initial lectin chromatography step and digested with endoproteinase Lys-C. Glycosylated peptides are then selected from the digest mixture by a second lectin chromatography step. The glycan components are removed with N-glycosidase F and the peptides digested with trypsin before analysis by on-line reversed-phase liquid chromatography mass spectrometry. Using two different lectins, concanavalin A and wheat germ agglutinin, this procedure was applied to human serum and a total of 86 N-glycosylation sites in 77 proteins were identified.     

Cystic fibrosis as a cause of infertility

Cystic fibrosis (CF) is one of the autosomal recessive diseases, caused by mutations in a gene known as cystic fibrosis transmembrane regulator (CFTR). The majority of adult males with CF (99%) is characterized by congenital bilateral absence of vas deferens (CBAVD). CBAVD is encountered in 1-2% of infertile males without CF. Females with CF are found to be less fertile than normal healthy women. In females with CF, delayed puberty and amenorrhoea are common due to malnutrition. CFTR mutations are also associated with congenital absence of the uterus and vagina (CAUV). The National Institutes of Health recommend genetic counseling for any couple seeking assisted reproductive techniques with a CF male or obstructive azoospermia which is positive for a CF mutation.     

Cooperative interactions of simian immunodeficiency virus Nef,AP-2, and CD3-zeta mediate the selective induction of T-cell receptor-CD3 endocytosis

The Nef proteins of human immunodeficiency virus and simian immunodeficiency virus (SIV) bind the AP-1 and AP-2 clathrin adaptors to downmodulate the expression of CD4 and CD28 by recruiting them to sites of AP-2 clathrin-dependent endocytosis. Additionally, SIV Nef directly binds the CD3-zeta subunit of the CD3 complex and downmodulates the T-cell receptor (TCR)-CD3 complex. We report here that SIV mac239 Nef induces the endocytosis of TCR-CD3 in Jurkat T cells. SIV Nef also induces the endocytosis of a chimeric CD8-CD3-zeta protein containing only the CD3-zeta cytoplasmic domain (8-zeta), in the absence of other CD3 subunits. Thus, the interaction of SIV Nef with CD3-zeta likely mediates the induction of TCR-CD3 endocytosis. In cells expressing SIV Nef and 8-zeta, both proteins colocalize with AP-2, indicating that Nef induces 8-zeta internalization via this pathway. Surprisingly, deletion of constitutively strong AP-2 binding determinants (CAIDs) in SIV Nef had little effect on its ability to induce TCR-CD3, or 8-zeta endocytosis, even though these determinants are required for the induction of CD4 and CD28 endocytosis via this pathway. Fluorescent microscopic analyses revealed that while neither the mutant SIV Nef protein nor 8-zeta colocalized with AP-2 when expressed independently, both proteins colocalized with AP-2 when coexpressed. In vitro binding studies using recombinant SIV Nef proteins lacking CAIDs and recombinant CD3-zeta cytoplasmic domain demonstrated that SIV Nef and CD3-zeta cooperate to bind AP-2 via a novel interaction. The fact that Nef uses distinct AP-2 interaction surfaces to recruit specific membrane receptors demonstrates how Nef independently selects distinct types of target receptors and recruits them to AP-2 for endocytosis.     

Alleviation of mutagenic effects of polycyclic aromatic agents (quinacrine mustard, ICR-191 and ICR-170) by caffeine and pentoxifylline

Previous studies performed by others indicated that apart from its other biological effects, caffeine (CAF) may have a role in protection of organisms against cancer. However, biological mechanism of this phenomenon remained unknown. Recent studies suggested that caffeine can form stacking (pi-pi) complexes with polycyclic aromatic chemicals. Therefore, one might speculate that effective concentrations of polycyclic aromatic mutagens could be reduced in the presence of caffeine. Here we demonstrate that caffeine and another xanthine, pentoxifylline (PTX), effectively alleviate mutagenic action of polycyclic aromatic agents (exemplified by quinacrine mustard (QM), 2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine.2HCl (ICR-191) and 1,3,7-propanediamine-N-(2-chloroethyl)-N'-(6-chloro-2-methoxy-9-acridinyl)-N-ethyl.2HCl (ICR-170)), but not of aliphatic mutagens (exemplified by mechlorethamine), in the recently developed mutagenicity test based on bacterium Vibrio harveyi. Biophysical studies indicated that caffeine and pentoxifylline can form stacking complexes with the aromatic agents mentioned above. Molecular modeling also confirmed a possibility of stacking interactions between examined molecules.     

Radical scavenging properties of genistein

The reactivity of genistein toward reactive radical species has been investigated by means of pulse radiolysis. The values of rate constants, respectively 2.3 x 10(10) M(-1)s(-1) and 1.3 x 10(10) M(-1)s(-1) for the reaction with hydroxyl radical at pH 8.3 and 3.0, are close to diffusion limit indicating that genistein is a potent hydroxyl radical scavenger. The reactivity of genistein towards one-electron oxidants has also been investigated. The rate constants k = 4.6 x 10(9) M(-1)s(-1) (pH 8.3) and 6.7 x 10(8) M(-1)s(-1) (pH 7.6) have been determined for the reaction of genistein with *N3 and Br2*- radicals, respectively. For both oxidants the rate constants at pH 3 does not exceed 10(8) M(-1)s(-1). The differences in reactivity of genistein towards the oxidants at different acidity of the solution have been assumed to arise from the acid-base equilibria of genistein. The dissociation constants for genistein (pKa: 7.2, 10.0, and 13.1) have been evaluated spectroscopically. The influence of acid-base equilibria on bond dissociation energy and ionization potential for genistein has also been investigated by means of DFT calculations. It has been concluded on the basis of these calculations that monoanionic form of genistein existing at physiological pH is more powerful radical scavenger than the neutral molecule.     

PDZ tandem of human syntenin: crystal structure and functional properties

Syntenin, a 33 kDa protein, interacts with several cell membrane receptors and with merlin, the product of the causal gene for neurofibromatosis type II. We report a crystal structure of the functional fragment of human syntenin containing two canonical PDZ domains, as well as binding studies for full-length syntenin, the PDZ tandem, and isolated PDZ domains. We show that the functional properties of syntenin are a result of independent interactions with target peptides, and that each domain is able to bind peptides belonging to two different classes: PDZ1 binds peptides from classes I and III, while PDZ2 interacts with classes I and II. The independent binding of merlin by PDZ1 and syndecan-4 by PDZ2 provides direct evidence for the coupling of syndecan-mediated signaling to actin regulation by merlin.     

Calcium-regulated interaction of Sgt1 with S100A6 (calcyclin) and other S100 proteins

S100A6 (calcyclin), a small calcium-binding protein from the S100 family, interacts with several target proteins in a calcium-regulated manner. One target is Calcyclin-Binding Protein/Siah-1-Interacting Protein (CacyBP/SIP), a component of a novel pathway of beta-catenin ubiquitination. A recently discovered yeast homolog of CacyBP/SIP, Sgt1, associates with Skp1 and regulates its function in the Skp1/Cullin1/F-box complex ubiquitin ligase and in kinetochore complexes. S100A6-binding domain of CacyBP/SIP is in its C-terminal region, where the homology between CacyBP/SIP and Sgt1 is the greatest. Therefore, we hypothesized that Sgt1, through its C-terminal region, interacts with S100A6. We tested this hypothesis by performing affinity chromatography and chemical cross-linking experiments. Our results showed that Sgt1 binds to S100A6 in a calcium-regulated manner and that the S100A6-binding domain in Sgt1 is comprised of 71 C-terminal residues. Moreover, S100A6 does not influence Skp1-Sgt1 binding, a result suggesting that separate Sgt1 domains are responsible for interactions with S100A6 and Skp1. Sgt1 binds not only to S100A6 but also to S100B and S100P, other members of the S100 family. The interaction between S100A6 and Sgt1 is likely to be physiologically relevant because both proteins were co-immunoprecipitated from HEp-2 cell line extract using monoclonal anti-S100A6 antibody. Phosphorylation of the S100A6-binding domain of Sgt1 by casein kinase II was inhibited by S100A6, a result suggesting that the role of S100A6 binding is to regulate the phosphorylation of Sgt1. These findings suggest that protein ubiquitination via Sgt1-dependent pathway can be regulated by S100 proteins.     

Structural consequences of accommodation of four non-cognate amino acid residues in the S1 pocket of bovine trypsin and chymotrypsin

Crystal structures of P1 Gly, Val, Leu and Phe bovine pancreatic trypsin inhibitor (BPTI) variants in complex with two serine proteinases, bovine trypsin and chymotrypsin, have been determined. The association constants for the four mutants with the two enzymes show that the enlargement of the volume of the P1 residue is accompanied by an increase of the binding energy, which is more pronounced for bovine chymotrypsin. Since the conformation of the P1 side-chains in the two S1 pockets is very similar, we suggest that the difference in DeltaG values between the enzymes must arise from the more polar environment of the S1 site of trypsin. This results mainly from the substitutions of Met192 and Ser189 observed in chymotrypsin with Gln192 and Asp189 present in trypsin. The more polar interior of the S1 site of trypsin is reflected by a much higher order of the solvent network in the empty pocket of the enzyme, as is observed in the complexes of the two enzymes with the P1 Gly BPTI variant. The more optimal binding of the large hydrophobic P1 residues by chymotrypsin is also reflected by shrinkage of the S1 pocket upon the accommodation of the cognate residues of this enzyme. Conversely, the S1 pocket of trypsin expands upon binding of such side-chains, possibly to avoid interaction with the polar residues of the walls. Further differentiation between the two enzymes is achieved by small differences in the shape of the S1 sites, resulting in an unequal steric hindrance of some of the side-chains, as observed for the gamma-branched P1 Leu variant of BPTI, which is much more favored by bovine chymotrypsin than trypsin. Analysis of the discrimination of beta-branched residues by trypsin and chymotrypsin is based on the complexes with the P1 Val BPTI variant. Steric repulsion of the P1 Val residue by the walls of the S1 pocket of both enzymes prevents the P1 Val side-chain from adopting the most optimal chi1 value.