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Human xylosyltransferase I and N-terminal truncated forms: functional characterization of the core enzyme.

ABSTRACT: Human XT-I (xylosyltransferase I; EC 2.4.2.26) initiates the biosynthesis of the glycosaminoglycan linkage region and is a diagnostic marker of an enhanced proteoglycan biosynthesis. In the present study, we have investigated mutant enzymes of human XT-I and assessed the impact of the N-terminal region on the enzymatic activity. Soluble mutant enzymes of human XT-I with deletions at the N-terminal domain were expressed in insect cells and analysed for catalytic activity. As many as 260 amino acids could be truncated at the N-terminal region of the enzyme without affecting its catalytic activity. However, truncation of 266, 272 and 273 amino acids resulted in a 70, 90 and >98% loss in catalytic activity. Interestingly, deletion of the single 12 amino acid motif G261KEAISALSRAK272 leads to a loss-of-function XT-I mutant. This is in agreement with our findings analysing the importance of the Cys residues where we have shown that C276A mutation resulted in a nearly inactive XT-I enzyme. Moreover, we investigated the location of the heparin-binding site of human XT-I using the truncated mutants. Heparin binding was observed to be slightly altered in mutants lacking 289 or 568 amino acids, but deletion of the potential heparin-binding motif P721KKVFKI727 did not lead to a loss of heparin binding capacity. The effect of heparin or UDP on the XT-I activity of all mutants was not significantly different from that of the wild-type. Our study demonstrates that over 80% of the nucleotide sequence of the XT-I-cDNA is necessary for expressing a recombinant enzyme with full catalytic activity.

SUBMITTER: Muller S

PROVIDER: S-EPMC1386014 | biostudies-literature | 2006 Feb

REPOSITORIES: biostudies-literature

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Human xylosyltransferase I and N-terminal truncated forms: functional characterization of the core enzyme.

Müller Sandra S Disse Jennifer J Schöttler Manuela M Schön Sylvia S Prante Christian C Brinkmann Thomas T Kuhn Joachim J Kleesiek Knut K Götting Christian C

The Biochemical journal 20060201 Pt 1

Human XT-I (xylosyltransferase I; EC 2.4.2.26) initiates the biosynthesis of the glycosaminoglycan linkage region and is a diagnostic marker of an enhanced proteoglycan biosynthesis. In the present study, we have investigated mutant enzymes of human XT-I and assessed the impact of the N-terminal region on the enzymatic activity. Soluble mutant enzymes of human XT-I with deletions at the N-terminal domain were expressed in insect cells and analysed for catalytic activity. As many as 260 amino aci ...[more]

PMID: 16225459

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Project description:UnlabelledThere is increasing evidence that many amyloids in living cells have physiological functions. On the surfaces of fungal cells, amyloid core sequences in adhesins can aggregate into 100- to 1,000-nm-wide patches to form high-avidity adhesion nanodomains on the cell surface. The nanodomains form through interactions that have amyloid-like properties: binding of amyloid dyes, perturbation by antiamyloid agents, and interaction with homologous sequences. To test whether these functional interactions are mediated by typical amyloid interactions, we substituted an amyloid core sequence, LVFFA, from human Aβ protein for the native sequence IVIVA in the 1,419-residue Candida albicans adhesin Als5p. The chimeric protein formed cell surface nanodomains and mediated cellular aggregation. The native sequence and chimeric adhesins responded similarly to the amyloid dye thioflavin T and to amyloid perturbants. However, unlike the native protein, the nanodomains formed by the chimeric protein were not force activated and formed less-robust aggregates under flow. These results showed the similarity of amyloid interactions in the amyloid core sequences of native Als5p and Aβ, but they also highlighted emergent properties of the native sequence. Also, a peptide composed of the Aβ amyloid sequence flanked by amino acids from the adhesin formed two-dimensional sheets with sizes similar to the cell surface patches of the adhesins. These results inform an initial model for the structure of fungal cell surface amyloid nanodomains.ImportanceProtein amyloid aggregates are markers of neurodegenerative diseases such as Alzheimer's and Parkinsonism. Nevertheless, there are also functional amyloids, including biofilm-associated amyloids in bacteria and fungi. In fungi, glycoprotein adhesins aggregate into cell surface patches through amyloid-like interactions, and the adhesin clustering strengthens cell-cell binding. These fungal surface amyloid nanodomains mediate biofilm persistence under flow, and they also moderate host inflammatory responses in fungal infections. To determine whether the amyloid-like properties of fungal surface nanodomains are sequence specific, we ask whether a disease-associated amyloid core sequence has properties equivalent to those of the native sequence in a fungal adhesin. A chimeric adhesin with an amyloid sequence from the Alzheimer's disease protein Aβ instead of its native sequence effectively clustered the adhesins on the cell surface, but it showed a different response to hydrodynamic shear. These results begin an analysis of the sequence dependence for newly discovered activities for fungal surface amyloid nanodomains.

Dataset Information

Human xylosyltransferase I and N-terminal truncated forms: functional characterization of the core enzyme.

Publications

Human xylosyltransferase I and N-terminal truncated forms: functional characterization of the core enzyme.

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