Magnus Axelsson

Dimerization of the Human MUC2 Mucin in the Endoplasmic Reticulum Is Followed by a N-Glycosylation-dependent Transfer of the Mono- and Dimers to the Golgi Apparatus

Pulse-chase experiments in the colon cell line LS 174T combined with subcellular fractionation by sucrose density gradient centrifugation showed that the initial dimerization of the MUC2 apomucin started directly after translocation of the apomucin into the rough endoplasmic reticulum as detected by calnexin reactivity. As the mono- and dimers were chased, O-glycosylated MUC2 mono- and dimers were precipitated using anO-glycosylation-insensitive antiserum against the N-terminal domain of the MUC2 mucin. These O-glycosylated species were precipitated from the fractions that comigrated with the galactosyltransferase activity during the subcellular fractionation, indicating that not only MUC2 dimers but also a significant amount of monomers are transferred into the Golgi apparatus. Inhibition ofN-glycosylation with tunicamycin treatment slowed down the rate of dimerization and introduced further oligomerization of the MUC2 apomucin in the endoplasmic reticulum. Results of two-dimensional gel electrophoresis demonstrated that these oligomers (putative tri- and tetramers) were stabilized by disulfide bonds. The non-N-glycosylated species of the MUC2 mucin were retained in the endoplasmic reticulum because no O-glycosylated species were precipitated after inhibition by tunicamycin. This suggests that N-glycans of MUC2 are necessary for the correct folding and dimerization of the MUC2 mucin.

O-Glycosylated MUC2 Monomer and Dimer from LS 174T Cells Are Water-soluble, whereas Larger MUC2 Species Formed Early during Biosynthesis Are Insoluble and Contain Nonreducible Intermolecular Bonds

The MUC2 mucin is the major gel-forming mucin in the small and large intestine. Due to its sequence similarities with the von Willebrand factor, it has been suggested to dimerize in the endoplasmic reticulum and polymerize in the trans-Golgi network. Using an O-glycosylation-sensitive MUC2 antiserum, a dimerization has been shown to occur in the endoplasmic reticulum of LS 174T cells (Asker, N., Axelsson, M. A. B., Olofsson, S.-O., and Hansson, G. C. (1998) J. Biol. Chem. 273, 18857–18863). Using an antiserum immunoprecipitating O-glycosylated MUC2 mucin, monomers and dimers were shown to occur in soluble form in the lysate of LS 174T cells. The amount of O-glycosylated dimer was small, and no larger species were found even after long chase periods. However, most of the labeled MUC2 mucin was found in pelleted debris of the cell lysate. This insoluble MUC2 mucin was recovered by immunoprecipitation after reduction of disulfide bonds. Analysis by agarose gel electrophoresis revealed two bands, of which the smaller migrated as the O-glycosylated monomer and the larger migrated as the O-glycosylated dimer of the cell lysis supernatant. Mucins insoluble in 6 m guanidinium chloride could also be obtained from LS 174T cells. Such mucins have earlier been found in the small intestine (Carlstedt, I., Herrmann, A., Karlsson, H., Sheehan, J., Fransson, L.-Å., and Hansson, G. C. (1993) J. Biol. Chem. 268, 18771–18781). Reduction of the mucins followed by purification by isopycnic density gradient ultracentrifugation and analysis by agarose gel electrophoresis revealed two bands reacting with an anti-MUC2 tandem repeat antibody after deglycosylation. These bands migrated identically to the bands shown by metabolic labeling, and they could also be separated by rate zonal ultracentrifugation. These results suggest that the MUC2 mucin is forming nonreducible intermolecular bonds early in biosynthesis, but after initial O-glycosylation.

Mapping of Residues in the NADP(H)-binding Site of Proton-translocating Nicotinamide Nucleotide Transhydrogenase fromEscherichia coli A STUDY OF STRUCTURE AND FUNCTION

Conformational changes in proton pumping transhydrogenases have been suggested to be dependent on binding of NADP(H) and the redox state of this substrate. Based on a detailed amino acid sequence analysis, it is argued that a classical βαβαβ dinucleotide binding fold is responsible for binding NADP(H). A model defining βA, αB, βB, βD, and βE of this domain is presented. To test this model, four single cysteine mutants (cfβA348C, cfβA390C, cfβK424C, and cfβR425C) were introduced into a functional cysteine-free transhydrogenase. Also, five cysteine mutants were constructed in the isolated domain III ofEscherichia coli transhydrogenase (ecIIIH345C, ecIIIA348C, ecIIIR350C, ecIIID392C, and ecIIIK424C). In addition to kinetic characterizations, effects of sulfhydryl-specific labeling withN-ethylmaleimide, 2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid, and diazotized 3-aminopyridine adenine dinucleotide (phosphate) were examined. The results are consistent with the view that, in agreement with the model, β-Ala348, β-Arg350, β-Ala390, β-Asp392, and β-Lys424 are located in or close to the NADP(H) site. More specifically, β-Ala348 succeeds βB. The remarkable reactivity of βR350C toward NNADP suggests that this residue is close to the nicotinamide moiety of NADP(H). β-Ala390 and β-Asp392 terminate or succeed βD, and are thus, together with the region following βA, creating the switch point crevice where NADP(H) binds. β-Asp392 is particularly important for the substrate affinity, but it could also have a more complex role in the coupling mechanism for transhydrogenase.

Deglycosylation by gaseous hydrogen fluoride of mucus glycoproteins immobilized on nylon membranes and in microtiter wells

Strongly reacting antibodies specific for defined mucin gene products are often directed against the mucin protein backbone of the heavily glycosylated serine/threonine rich regions. A prerequisite for the use of such antibodies is often the complete removal of the oligosaccharides from the protein. This paper describes an efficient one-step deglycosylation method using gaseous hydrogen fluoride on nylon blotting membranes and microtiter wells.