Distinctive and Complementary MS2 Fragmentation Characteristics for Identification of Sulfated Sialylated N-Glycopeptides by nanoLC-MS/MS Workflow.

High sensitivity identification of sulfated glycans carried on specific sites of glycoproteins is an important requisite for investigation of molecular recognition events involved in diverse biological processes. However, aiming for resolving site-specific glycosylation of sulfated glycopeptides by direct LC-MS2 sequencing is technically most challenging. Other than the usual limiting factors such as lower abundance and ionization efficiency compared to analysis of non-glycosylated peptides, confident identification of sulfated glycopeptides among the more abundant non-sulfated glycopeptides requires additional considerations in the selective enrichment and detection strategies. Metal oxide has been applied to enrich phosphopeptides and sialylated glycopeptides, but its use to capture sulfated glycopeptides has not been investigated. Likewise, various complementary MS2 fragmentation modes have yet to be tested against sialylated and non-sialylated sulfoglycopeptides due to limited appropriate sample availability. In this study, we have investigated the feasibility of sequencing tryptic sulfated N-glycopeptide and its MS2 fragmentation characteristics by first optimizing the enrichment methods to allow efficient LC-MS detection and MS2 analysis by a combination of CID, HCD, ETD, and EThcD on hybrid and tribrid Orbitrap instruments. Characteristic sulfated glyco-oxonium ions and direct loss of sulfite from precursors were detected as evidences of sulfate modification. It is anticipated that the technical advances demonstrated in this study would allow a feasible extension of our sulfoglycomic analysis to sulfoglycoproteomics. Graphical Abstract ᅟ.

Investigations into the development of catalytic activity in anti-acetylcholinesterase idiotypic and anti-idiotypic antibodies.

We have previously described anti-acetylcholinesterase antibodies that display acetylcholinesterase-like catalytic activity. No evidence of contaminating enzymes was found, and the antibodies are kinetically and apparently structurally distinct from both acetylcholinesterase (AChE) and butyrylcholinesterase. We have also mimicked the antibody catalytic sites in anti-anti-idiotypic (Ab3) antibodies. Independently from us, similar acetylcholinesterase-like antibodies have been raised as anti-idiotypic (Ab2) antibodies against a non-catalytic anti-acetylcholinesterase antibody, AE-2. In this paper, we describe an epitope analysis, using synthetic peptides in ELISA and competition ELISA, and a peptide array, of five catalytic anti-acetylcholinesterase antibodies (Ab1s), three catalytic Ab3s, as well as antibody AE-2 and a non-catalytic Ab2. The catalytic Ab1s and Ab3s recognized three Pro- and Gly-containing sequences ((40)PPMGPRRFL, (78)PGFEGTE, and (258)PPGGTGGNDTELVAC) on the AChE surface. As these sequences do not adjoin in the AChE structure, recognition would appear to be due to cross-reaction. This was confirmed by the observation that the sequences superimpose structurally. The non-catalytic antibodies, AE-2 and the Ab2, recognized AChE's peripheral anionic site (PAS), in particular, the sequence (70)YQYVD, which contains two of the site's residues. The crystal structure of the AChE tetramer (Bourne et al., 1999) shows direct interaction and high complementarity between the (257)CPPGGTGGNDTELVAC sequence and the PAS. Antibodies recognizing the sequence and the PAS may, in turn, be complementary; this may account for the apparent paradox of catalytic development in both Ab1s and Ab2s. The PAS binds, but does not hydrolyze, substrate. The catalytic Ab1s, therefore, recognize a site that may function as a substrate analog, and this, together with the presence of an Arg-Glu salt bridge in the epitope, suggests mechanisms whereby catalytic activity may have developed. In conclusion, the development of AChE-like catalytic activity in anti-AChE Ab1s and Ab2s appears to be the result of a combination of structural complementarity to a substrate-binding site, charge complementarity to a salt bridge, and specific structural peculiarities of the AChE molecule.

Mixed-disulfide folding intermediates between thyroglobulin and endoplasmic reticulum resident oxidoreductases ERp57 and protein disulfide isomerase.

We present the first identification of transient folding intermediates of endogenous thyroglobulin (Tg; a large homodimeric secretory glycoprotein of thyrocytes), which include mixed disulfides with endogenous oxidoreductases servicing Tg folding needs. Formation of disulfide-linked Tg adducts with endoplasmic reticulum (ER) oxidoreductases begins cotranslationally. Inhibition of ER glucosidase activity blocked formation of a subgroup of Tg adducts containing ERp57 while causing increased Tg adduct formation with protein disulfide isomerase (PDI), delayed adduct resolution, perturbed oxidative folding of Tg monomers, impaired Tg dimerization, increased Tg association with BiP/GRP78 and GRP94, activation of the unfolded protein response, increased ER-associated degradation of a subpopulation of Tg, partial Tg escape from ER quality control with increased secretion of free monomers, and decreased overall Tg secretion. These data point towards mixed disulfides with the ERp57 oxidoreductase in conjunction with calreticulin/calnexin chaperones acting as normal early Tg folding intermediates that can be "substituted" by PDI adducts only at the expense of lower folding efficiency with resultant ER stress.

The acetylcholinesterase homology region is essential for normal conformational maturation and secretion of thyroglobulin.

Secretion of thyroglobulin (Tg, a large homodimeric glycoprotein) is essential to deliver Tg to its site of iodination for thyroxine biosynthesis. An L2263P missense mutation in Tg has been proposed as the molecular defect causing congenital goitrous hypothyroidism in cog/cog mice due to perturbed Tg homodimerization, resulting in its retention within the endoplasmic reticulum. The mutation falls within a carboxyl-terminal region of Tg with high structural similarity to the entirety of acetylcholinesterase (AChE), a secretory protein that also forms homodimers. We provide new evidence that authentic AChE and the cholinesterase-like domain of Tg share a common tertiary structure. Moreover, we find that a Tg truncation, deleted of the cholinesterase-like region (but not a comparably sized deletion of internal Tg regions), blocks Tg export. Appending to this truncation a cDNA encoding authentic AChE results in translation of a chimeric protein in which AChE is present in a native, enzymatically active (albeit latent) conformation, and this fully rescues Tg secretion. Introduction of the cog mutation inhibits AChE enzyme activity, and established denaturing mutations of AChE block secretion of the Tg. Additional studies show that the native structure of the AChE region functions as a "dimerization domain," facilitating intracellular transport of Tg to the site of thyroid hormonogenesis.

Molecular characterization of a new lectin from the marine alga Ulva pertusa.

A new lectin, named UPL1, was purified from a green alga Ulva pertusa by an affinity chromatography on the bovine-thyroglobulin-Sepharose 4B column. The molecular mass of the algal lectin was about 23 kD by SDS-PAGE, and it specifically agglutinated rabbit erythrocytes. The hemagglutinating activity for rabbit erythrocytes could be inhibited by bovine thyroglobulin and N-acetyl-D-glucosamine. The lectin UPL1 required divalent cations for maintenance of its biological activity, and was heat-stable, and had higher activity within pH 6-8. The N-terminal amino acid sequence of the purified lectin was determined (P83209) and a set of degenerate primers were designed. The full-length cDNA of the lectin was cloned by rapid amplification of cDNA ends (RACE) method (AY433960). Sequence analysis of upl1 indicated it was 1084 bp long, and encoded a premature protein of 203 amino acids. The N-terminal sequence of the mature UPL1 polypeptide started at amino acid 54 of the deduced sequence from the cDNA, indicating 53 amino acids lost due to posttranslational modification. The primary structure of the Ulva pertusa lectin did not show amino acid sequence similarity with known plant and animal lectins. Hence, this protein may be the paradigm of a novel lectin family.

A single amino acid change in the acetylcholinesterase-like domain of thyroglobulin causes congenital goiter with hypothyroidism in the cog/cog mouse: a model of human endoplasmic reticulum storage diseases.

Newly synthesized thyroglobulin (Tg), the major secretory glycoprotein of the thyroid gland, folds and homodimerizes in the endoplasmic reticulum (ER) before its export to the site of iodination, where it serves as the precursor for thyroid hormone synthesis. In families with defective Tg export, affected individuals suffer from a thyroidal ER storage disease characterized by a distended thyrocyte ER containing misfolded Tg, along with induced ER molecular chaperones. Inherited as an autosomal recessive trait, deficient Tg causes congenital hypothyroidism in newborns that, if untreated, results in goiter along with serious cognitive and growth defects. Recently, a similar phenotype has been observed in inbred cog/cog mice, although the precise molecular defect has remained undefined. Here, we have isolated and cloned a full-length 8.5-kb Tg cDNA from cog/cog mice and unaffected isogenic AKR/J mice. Comparison of the complete sequences reveals that cog/cog mice express a Leu-2263 --> Pro missense mutation in the acetylcholinesterase-homology domain of Tg. Heterologous expression studies in COS cells indicate that cog Tg exhibits a severe defect in exit from the ER. Site-directed mutagenesis of cog Tg to convert the single amino acid back to Leu-2263 restores normal Tg secretion. We conclude that the cog mutation in Tg is responsible for this ER storage disease that causes thyroid dyshormonogenesis.

Iodine content of rat thyroglobulin affects its antigenicity in inducing lymphocytic thyroiditis in the BB/Wor rat.

The BB/Wor rat develops spontaneous insulin dependent diabetes mellitus (DM) and lymphocytic thyroiditis (LT). We have recently demonstrated that immunization of BB/Wor rats with allogeneic thyroglobulin (Tg) induces LT at an early age. The incidence of spontaneous and Tg induced LT is extremely variable among different BB/Wor sublines. It has been shown that high iodine diet significantly increases the incidence of spontaneous lymphocytic thyroiditis (LT) and low iodine diet significantly decreases the incidence of LT in genetically predisposed BB/Wor rats. Recent studies on thyroglobulin (Tg) induced LT in chicken and mouse have shown that iodine rich Tg is far more antigenic than Tg with a low iodine content, suggesting that a high iodine diet increases the immunogenicity of Tg molecule. In order to determine whether the extent of Tg iodination would affect its immunogenicity in the BB/Wor rats, the current study was carried out. Normal iodine Tg (NTg) or low iodine Tg (LTg) was obtained from thyroids of rats that were placed on regular diet or regular diet plus 0.5% methimazole, respectively. 120 rats from the NB (highly susceptible) and BB (low susceptible) sublines were randomized in three groups. Immunization was carried out with a 1:1 emulsion of complete Freund's adjuvant (CFA) and LTg, NTg (0.6 mg/rat) or saline at 30 and 37 days of age. Since spontaneous LT rarely occurs before age 75 days, rats were sacrificed at age 65 days to specifically study Tg induced LT. Immunization with NTg induced LT in 31% of the NB rats, but not in the BB subline. LTg did not induce LT in either subline.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of thyrotropin- and dibutyryl cyclic AMP- induced thyroglobulin iodination in cultured human thyroid cells.

The relative degree of 125-I labelling of thyroglobulin-- bound mono-iodotyrosine (MIT) and di-iodotyrosine (DIT) in isolated, cultured human thyroid cells has been compared following exposure of 125-I supplemented cells to 100 mU/ml of bovine thyrotropin (TSH) or 1.0 mM dibutyryl cyclic AMP (dBcAMP) for 96 hours. Pronase digestion of the lysed cells and Sephadex G-10 fractionation of the digested lysates revealed a predominance of [125-I]MIT over [125-I]DIT in both sets of experimental cells as well as in controls. Levels of [125-I]DIT, however, were only enhanced above control values in cells incubated with TSH. These findings suggest that an increase in availability of intracellular iodide, following cellular exposure to TSH, may facilitate a preferential synthesis of DIT relative to that of MIT. This theory offers an explanation for the differential effects of TSH and dibutyryl cyclic AMP on the levels of newly--synthesised T4 recovered from the cells used in this study, and from the culture medium in a previous investigation.