Recognition signal for C-mannosylation of Trp-7 in RNase 2 consists of sequence Trp-x-x-Trp - PubMed (original) (raw)

Recognition signal for C-mannosylation of Trp-7 in RNase 2 consists of sequence Trp-x-x-Trp

J Krieg et al. Mol Biol Cell. 1998 Feb.

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Abstract

C2-alpha-Mannosyltryptophan was discovered in human RNase 2, an enzyme that occurs in eosinophils and is involved in host defense. It represents a novel way of attaching carbohydrate to a protein in addition to the well-known N- and O-glycosylations. The reaction is specific, as in RNase 2 Trp-7, but never Trp-10, which is modified. In this article, we address which structural features provide the specificity of the reaction. Expression of chimeras of RNase 2 and nonglycosylated RNase 4 and deletion mutants in HEK293 cells identified residues 1-13 to be sufficient for C-mannosylation. Site-directed mutagenesis revealed the sequence Trp-x-x-Trp, in which the first Trp becomes mannosylated, as the specificity determinant. The Trp residue at position +3 can be replaced by Phe, which reduces the efficiency of the reaction threefold. Interpretation of the data in the context of the three-dimensional structure of RNase 2 strongly suggests that the primary, rather than the tertiary, structure forms the determinant. The sequence motif occurs in 336 mammalian proteins currently present in protein databases. Two of these proteins were analyzed protein chemically, which showed partial C-glycosylation of recombinant human interleukin 12. The frequent occurrence of the protein recognition motif suggests that C-glycosides could be part of the structure of more proteins than assumed so far.

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Figures

Figure 1

Figure 1

Structure of hybrid RNases. (A) The RNase 2 portion of a hybrid is depicted as an open rectangle, whereas that of RNase 4 (numbering in italic) is hatched. Trp-7 has been underlined. (B) Comparison of the primary structures of human RNase 2 and porcine RNase 4. Amino acids in common are indicated by a line when they occur at the surface of the protein and by a colon when they are buried.

Figure 2

Figure 2

Reversed-phase HPLC purification of RNase 2.4. Immunopurified RNase 2.4 was chromatographed on a C4 column (1 mm in diameter) equilibrated in 0.1% TFA. A linear gradient of 0–80% solvent B (70% CH3CN in 0.085% TFA) over 75 min was used at a flow rate of 50 μl/min. The inset shows a Western blot analysis of the fractions using antibodies against RNase 4 or modification-specific antibodies, α(5–10).

Figure 3

Figure 3

Western blot analysis of single-site mutants of RNase 2.4. Approximately 0.5 μg of each protein was electrophoresed on a 15% SDS-PAA gel and blotted onto nitrocellulose. The blots were probed with modification-specific antibodies, α(5–10), and, after stripping, with αRNase 4 antibodies.

Figure 4

Figure 4

Characterization of RNase 2.4 and its mutants. (A) Five micrograms of reduced and carboxymethylated RNase were digested at glutamic acid residues with endoproteinase Glu-C and fractionated by C8 reversed-phase LC-ESIMS. A 1-mm diameter column equilibrated in 95% solvent A (2% CH3CN, 0.05% TFA) and 5% solvent B (80% CH3CN, 0.045% TFA) was used. Peptides were eluted with a linear gradient of 5–40% solvent B at a flow rate of 50 μl/min. C-mannosylated (“m”) and unmodified (“u”) fragment −4 to 12 were assigned based on their molecular masses. The results obtained with wild-type RNase 2.4 (upper panel), mutant T6A (middle panel), and W10A (lower panel) are shown as representative examples. (B) Peptide −4 to 12 was digested with elastase and fractionated by reversed-phase LC-ESIMS. The peptide map obtained from the T6A mutant is shown as a representative example. Peptide 9–12 with unmodified Trp-10, 608 Da; peptide −4 to 8 with C-mannosylated Trp-7, 1542 Da. (C) ESIMS of the fragment −4 to 12 from RNase 2.4, W10A. The molecular mass of 1885 Da corresponds to that of the peptide with unmodified Trp.

Figure 5

Figure 5

Mutational analysis of RNase 2.4 and RNase 2. (A) The degree of C-mannosylation of Trp-7 in the indicated mutants of hybrid RNase 2.4 was determined from the ratio of modified and unmodified fragments −4 to 12 and is depicted by filled bars. (B) The degree of modification of Trp-7 in the indicated mutants of RNase 2 was determined as in A and are plotted as open bars for the experiment performed in 3T3 cells and as filled bars for the experiment performed in HEK293 cells. The data represent the average of at least two independent experiments. The SD was 1–16% of the mean.

Figure 6

Figure 6

Three-dimensional structure around the C-mannosylation site of recombinant RNase 2. The indole moieties of Trp-7 and -10 are shown in dark gray. Since the protein was produced in E. coli, Trp-7 is not C-mannosylated. The coordinates used to produce this figure were obtained from Mosimann et al., 1996.

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