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, 342 (6163), 1235-9

HCF-1 Is Cleaved in the Active Site of O-GlcNAc Transferase

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HCF-1 Is Cleaved in the Active Site of O-GlcNAc Transferase

Michael B Lazarus et al. Science.

Abstract

Host cell factor-1 (HCF-1), a transcriptional co-regulator of human cell-cycle progression, undergoes proteolytic maturation in which any of six repeated sequences is cleaved by the nutrient-responsive glycosyltransferase, O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT). We report that the tetratricopeptide-repeat domain of O-GlcNAc transferase binds the carboxyl-terminal portion of an HCF-1 proteolytic repeat such that the cleavage region lies in the glycosyltransferase active site above uridine diphosphate-GlcNAc. The conformation is similar to that of a glycosylation-competent peptide substrate. Cleavage occurs between cysteine and glutamate residues and results in a pyroglutamate product. Conversion of the cleavage site glutamate into serine converts an HCF-1 proteolytic repeat into a glycosylation substrate. Thus, protein glycosylation and HCF-1 cleavage occur in the same active site.

Figures

Fig. 1
Fig. 1. Effect of mutations in OGT and HCF-1 constructs on cleavage and glycosylation
(A) Top. Schematic of HCF-1 showing the six proteolytic repeats (rep1-rep6) with the amino acid identities of a representative pro repeat shown. The conserved residues are shown in yellow, with the E10 glutamate essential for cleavage shown in red. The repeats are subdivided into cleavage and threonine-rich regions(12). Site-specific proteolysis by OGT leads to the formation of HCF-1N and HCF-1C subunits. Bottom. Schematic of HCF-1 constructs used in this study. GST–HCF-1rep1 contains the first HCF-1PRO repeat and surrounding sequences fused to GST. Several S/T glycosylation sites are found in the HCF-1rep1 construct as schematized. The HCF3R construct contains only the first three HCF-1PRO repeats fused to an N-terminal His-tag. (B) Comparative cleavage and glycosylation activities of WT OGT and several catalytic domain mutants. GST–HCF-1rep1 was incubated in the absence (lane 1) or presence of WT OGT (lane 2) or the indicated mutants (lanes 3–5). HCF-1rep1 cleavage was detected by western blot analysis with anti-GST antibody and HCF-1rep1 glycosylation was detected with anti-O-GlcNAc antibody (RL2). (C) Cleavage activities of WT and mutant HCF-1rep1 constructs. WT GST–HCF1rep1 (lanes 1 and 2) or a threonine-rich region mutant (T17-22A; lanes 3–4) or the indicated E10 cleavage site mutants (E10A, E10Q, E10D, E10S; lanes 5–12) were incubated in the absence (–) or presence (+) of WT OGT as described in Materials and Methods. Cleavage was detected as in (B).
Fig. 2
Fig. 2. HCF-1PRO-repeat cleavage results in formation of a pyroglutamate product
(A) Cleavage of HCF3R requires UDP-GlcNAc. HCF3R was incubated with WT OGT (lanes 2–6), with K842A OGT (lane 7) or with OGT treated with a previously reported(18) covalent inhibitor BZX2 (lane 8), in the presence of UDP (lane 3), UDP-GlcNAc (lanes 4, 5, 7, 8), or UDP-5SGlcNAc (5S, lane 6). Alkaline phosphatase (AP) was added to some reactions, as indicated. Cleavage products were separated by SDS-PAGE and stained with Coomassie Blue. (B) LC-MS analysis of untreated HCF3R (black) and HCF3R cleavage products (red) following incubation with OGT and UDP-GlcNAc shows unexpected mass peaks. Detected and predicted MS peaks for different cleavage products are tabulated. (C) Mutation of E10 to alanine in the cleavage region of the second and third HCF-1PRO repeats produces a construct, HCF3R-EAA, containing only a single cleavable repeat. Pyroglutamate (pyroGlu) aminopeptidase removed a 111 Da fragment from the HCF3R-EAA C-terminal cleavage product, confirming the formation of pyroglutamate in the cleavage reaction.
Fig. 3
Fig. 3. The threonine-rich region of the HCF-1PRO repeat binds in the channel formed by the TPR domain of OGT
(A) Overall structure of the OGT:UDP:HCF-111–26 peptide complex. A 16-residue peptide comprising the threonine-rich region of HCF-1PRO repeat 2 (THETGTTNTATTATSN) was co-crystallized with UDP and a previously described(13) N-terminally truncated OGT construct (hOGT4.5) and refined to 1.8 Å. The OGT catalytic domain (red) and TPR domain (grey) along with the HCF-111–26 peptide (cyan) and UDP (yellow) are shown. (B) Close-up view of OGT-peptide interactions. The electron density around the visible portion of HCF-111–26 is shown as an FO-FC difference map contoured at 3σ. The peptide is shown in cyan. OGT sidechains that contact the peptide backbone are shown in yellow and OGT sidechains that contact HCF-1 peptide sidechains are shown in magenta. (C) Schematic of contacts between OGT sidechains and the threonine-rich region of the HCF-1PRO repeat 2 from the complex of OGT:UDP:HCF-1-E10A1–26. OGT sidechains are numbered and colored as in panel (B). (D) Mutations in the TPR domain of OGT (5N-5A) inhibit cleavage. Cleavage and glycosylation of GST–HCF-1rep1 were assayed, as in Figure 1C, in the absence (lane 1) or presence (lane 2) of WT OGT or the 5N-5A TPR-domain mutant in which Asn residues 322, 356, 390, 424 and 458 are mutated to alanine (lane 3). (E) OGT does not bind effectively in vitro to an HCF-1PRO repeat mutant containing mutations in the threonine-rich region (T17–22A). WT (lane 2) and mutant (lanes 1, 3 and 4) GST–HCF-1rep1 substrates were tested for OGT binding in the presence of UDP-GlcNAc using an OGT-directed pull-down assay. Anti-GST and anti-T7 antibodies were used to detect GST–HCF-1rep1 (upper panel) and OGT (lower panel), respectively, by western blotting.
Fig. 4
Fig. 4. HCF-1 cleavage takes place in the glycosyltransferase active site of OGT
(A) Overall structure of the OGT:UDP-5SGlcNAc:HCF-1-E10Q1–26 complex. The HCF-1 peptide is shown as spheres in cyan with the UDP-5SGlcNAc in yellow. (B) Close-up view of the two substrate analogs shown in yellow in the OGT active site. The entire cleavage region can be seen and the C-E10Q-T residues are annotated. The anomeric carbon of UDP-5SGlcNAc is indicated (C1). (C) Overlay of the substrate analogs from the OGT:UDP-5SGlcNAc:HCF-1 peptide complex (yellow) and the previously reported OGT:UDP-5SGlcNAc:CKIIA complex (cyan). CKII is a well-characterized OGT glycosylation substrate. The E10Q sidechain of the HCF-1 peptide is shown as transparent just after the β-carbon. (D) Mutating E10 to S in an HCF-1PRO repeat converts a cleavage substrate (HCF3R-EAA) into a glycosylation substrate (HCF-SAA), which is defective in cleavage. (Left panel) Cleavage products of HCF3R-EAA and HCF3R-SAA were separated by SDS-PAGE and stained with Coomassie Blue. (Right panel) Glycosylation of wild-type and mutant HCF3R substrates was carried out with 14C-UDP-GlcNAc and analyzed by PAGE. Full gels are shown fig. S10.

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