Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to multidimensional protein identification technology.

In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases.

The proteomes of human parotid and submandibular/sublingual gland salivas collected as the ductal secretions.

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.

Transport pathways for clearance of human Alzheimer's amyloid beta-peptide and apolipoproteins E and J in the mouse central nervous system.

Amyloid beta-peptide (Abeta) clearance from the central nervous system (CNS) maintains its low levels in brain. In Alzheimer's disease, Abeta accumulates in brain possibly because of its faulty CNS clearance and a deficient efflux across the blood-brain barrier (BBB). By using human-specific enzyme-linked immunosorbent assays, we measured a rapid 30 mins efflux at the BBB and transport via the interstitial fluid (ISF) bulk flow of human-unlabeled Abeta and of Abeta transport proteins, apolipoprotein E (apoE) and apoJ in mice. We show (i) Abeta40 is cleared rapidly across the BBB via low-density lipoprotein receptor-related protein (LRP)1 at a rate of 0.21 pmol/min g ISF or 6-fold faster than via the ISF flow; (ii) Abeta42 is removed across the BBB at a rate 1.9-fold slower compared with Abeta40; (iii) apoE, lipid-poor isoform 3, is cleared slowly via the ISF flow and across the BBB (0.03-0.04 pmol/min g ISF), and after lipidation its transport at the BBB becomes barely detectable within 30 mins; (iv) apoJ is eliminated rapidly across the BBB (0.16 pmol/min g ISF) via LRP2. Clearance rates of unlabeled and corresponding 125I-labeled Abeta and apolipoproteins were almost identical, but could not be measured at low physiologic levels by mass spectrometry. Amyloid beta-peptide 40 binding to apoE3 reduced its efflux rate at the BBB by 5.7-fold, whereas Abeta42 binding to apoJ enhanced Abeta42 BBB clearance rate by 83%. Thus, Abeta, apoE, and apoJ are cleared from brain by different transport pathways, and apoE and apoJ may critically modify Abeta clearance at the BBB.

Development of an oral biosensor for salivary amylase using a monodispersed silver for signal amplification.

An amperometric biosensor for monitoring the level of protein amylase in human saliva is described. A novel design and the preparation of amylase antibodies and antigens, essential for the development of the biosensor, are reported. The biosensor sensing elements comprise a layer of salivary antibody (or antigen) self-assembled onto Au-electrode via covalent attachment. Molecular recognition between the immobilized antibody and the salivary amylase proteins was monitored via an electroactive indicator (e.g., K(3)Fe(CN)(6)) or a monodispersed silver layer present in solution or electrochemically deposited onto the solid electrode. This electroactive indicator was oxidized or reduced and the resulting current change provided the analytical information about the concentration of the salivary proteins. The limit of detection of 1.57 pg/ml was obtained, in comparison to detection limits of 4.95 pg/ml obtained using potassium ferrocyanide as the redox probe and 10 ng/ml obtained using enzyme-linked immunosorbent assay. Cross-reactivity was tested against cystatin antibodies and was found to be less than 2.26%.

Nat3p and Mdm20p are required for function of yeast NatB Nalpha-terminal acetyltransferase and of actin and tropomyosin.

NatB Nalpha-terminal acetyltransferase of Saccharomyces cerevisiae acts cotranslationally on proteins with Met-Glu- or Met-Asp- termini and subclasses of proteins with Met-Asn- and Met-Met- termini. NatB is composed of the interacting Nat3p and Mdm20p subunits, both of which are required for acetyltransferase activity. The phenotypes of nat3-Delta and mdm20-Delta mutants are identical or nearly the same and include the following: diminished growth at elevated temperatures and on hyperosmotic and nonfermentable media; diminished mating; defective actin cables formation; abnormal mitochondrial and vacuolar inheritance; inhibition of growth by DNA-damaging agents such as methyl methanesulfonate, bleomycin, camptothecin, and hydroxyurea; and inhibition of growth by the antimitotic drugs benomyl and thiabendazole. The similarity of these phenotypes to the phenotypes of certain act1 and tpm1 mutants suggests that such multiple defects are caused by the lack of acetylation of actin and tropomyosins. However, the lack of acetylation of other unidentified proteins conceivably could cause the same phenotypes. Furthermore, unacetylated actin and certain N-terminally altered actins have comparable defective properties in vitro, particularly actin-activated ATPase activity and sliding velocity.

Stabilization of a pH-sensitive apoptosis-linked coiled coil through single point mutations.

The apoptosis-associated Par-4 protein has been implicated in cancers of the prostate, colon, and kidney, and in Alzheimer's and Huntington's diseases, among other neurodegenerative disorders. Previously, we have shown that a peptide from the Par-4 C-terminus, which is responsible for Par-4 self-association as well as interaction with all currently identified effector molecules, is natively unfolded at neutral pH, but forms a tightly associated coiled coil at acidic pH and low temperature. Here, we have alternately mutated the two acidic residues predicted to participate in repulsive electrostatic interactions at the coiled coil interhelical interface. Analysis of circular dichroism spectra reveals that a dramatic alteration of the folding/unfolding equilibrium of this peptide can be effected through directed-point mutagenesis, confirming that the two acidic residues are indeed key to the pH-dependent folding behavior of the Par-4 coiled coil, and further suggesting that alleviation of charge repulsion through exposure to either a low pH microenvironment or an electrostatically complementary environment may be necessary for efficient folding of the Par-4 C-terminus.

Bacterial protease treatment of natural rubber latex alters its primary immunogenicity in a mouse model of sensitization.

The purpose of the present investigation was to determine whether enzyme-treated (ET)-NRL is less immunogenic than untreated NRL in a BALB/c mouse model of primary in vivo sensitization following repeated subcutaneous injections with the aqueous phase of ammoniated NRL or ET-NRL. Mice immunized with NRL produced IgE against NRL and ET-NRL, indicating that protease treatment did not completely destroy IgE antibody epitopes. In contrast, ET-NRL-immunized mice did not produce IgE against either NRL or ET-NRL, suggesting that enzyme treatment reduced the number of antigenic polypeptides associated with NRL below the threshold for sensitization. Thelper-lymphocytes from NRL-immunized mice proliferated and produced IL-4 when stimulated in vitro with polypeptides from NRL, but not ET-NRL. In contrast, Thelper-lymphocytes from ET-NRL-immunized mice were nonresponsive to ET-NRL or NRL. We conclude that lack of IgE production by ET-NRL-immunized mice is likely related to a lack of T-cell help in the form of IL-4, rather than enzyme digestion of IgE antibody epitopes. These data indicate that there is an immunologic rationale for production of enzyme-treated NRL-containing medical devices.