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A Unique Molecular Chaperone Cosmc Required for Activity of the Mammalian Core 1 ␤3-Galactosyltransferase

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A Unique Molecular Chaperone Cosmc Required for Activity of the Mammalian Core 1 ␤3-Galactosyltransferase A unique molecular chaperone Cosmc required for activity of the mammalian core 1 ␤3-galactosyltransferase Tongzhong Ju and Richard D. Cummings* Department of Biochemistry and Molecular Biology and the Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104 Edited by Stuart A. Kornfeld, Washington University School of Medicine, St. Louis, MO, and approved October 23, 2002 (received for review July 23, 2002) Human core 1 ␤3-galactosyltransferase (C1␤3Gal-T) generates the to synthesize core 1 O-glycan is a potentially contributing factor core 1 O-glycan Gal␤1-3GalNAc␣1-Ser͞Thr (T antigen), which is a to several autoimmune diseases, including IgA nephropathy (9), precursor for many extended O-glycans in animal glycoproteins. Tn-syndrome (10), and Henoch-Scho¨nlein purpura (11). We report here that C1␤3Gal-T activity requires expression of a Intriguingly, the human T leukemic cell line Jurkat cells lack molecular chaperone designated Cosmc (core 1 ␤3-Gal-T-specific C1␤3Gal-T activity, lack core 1 O-glycans, and generate the Tn molecular chaperone). The human Cosmc gene is X-linked (Xq23), antigen (12, 13). Thus, an alteration in the expression of the and its cDNA predicts a 318-aa transmembrane protein (Ϸ36.4 kDa) C1␤3Gal-T is predicted to cause global changes in O-glycan with type II membrane topology. The human lymphoblastoid T cell structures in multiple glycoproteins. In exploring the factors that line Jurkat, which lacks C1␤3Gal-T activity and expresses the Tn regulate C1␤3Gal-T activity in Jurkat cells, we identified a antigen GalNAc␣1-Ser͞Thr, contains a normal gene and mRNA protein that associates with C1␤3Gal-T and is required for its encoding C1␤3Gal-T, but contains a mutated Cosmc with a deletion activity. We have designated this protein, which has the prop- introducing a premature stop codon. Expression of Cosmc cDNA in erties of a chaperone, Cosmc (core 1 ␤3-Gal-T-specific molec- Jurkat cells restored C1␤3Gal-T activity and T antigen expression. ular chaperone). Here, we show that a mutation in Cosmc in Without Cosmc, the C1␤3Gal-T is targeted to proteasomes. Expres- Jurkat cells causes loss of C1␤3Gal-T activity and targeting of sion of active C1␤3Gal-T in Hi-5 insect cells requires coexpression the inactive protein to the proteasome. Thus, mutations in of Cosmc. Overexpression of active C1␤3Gal-T in mammalian cell Cosmc may contribute to expression of Tn antigen in tumor cells lines also requires coexpression of Cosmc, indicating that endog- and human autoimmune diseases. enous Cosmc may be limiting. A small portion of C1␤3Gal-T copurifies with Cosmc from cell extracts, demonstrating physical Materials and Methods association of the proteins. These results indicate that Cosmc acts RT-PCR, PCR, Cloning of the RT-PCR Product, and Sequencing. Total as a specific molecular chaperone in assisting the folding͞stability RNA and genomic DNA from 5 ϫ 107 cells of Jurkat (clone E6-1, of C1␤3Gal-T. The identification of Cosmc, a uniquely specific ATCC TIB 152), Molt-4, and K562 cells were isolated by using molecular chaperone required for a glycosyltransferase expression the total RNA isolation kit (The RNA Company, Austin, TX) in mammalian cells, may shed light on the molecular basis of and QIAgen DNA mini kit (Qiagen, Valencia, CA). For RT- acquired human diseases involving altered O-glycosylation, such as PCR of Cosmc, the forward primer was 5Ј-CTCCATAGAG- IgA nephropathy, Tn syndrome, Henoch-Scho¨nlein purpura, and GAGTTGTTGC-3Ј, and the reverse primer was 5Ј- malignant transformation, all of which are associated with a TCACGCTTTTCTACCACTTC-3Ј. RT-PCR was performed in deficiency of C1␤3Gal-T activity. one step in 25 ␮l of reaction containing 500 ng of total RNA, RT͞Tag mix, and primers (SuperScript One-Step RT-PCR kit, he O-glycans in human glycoproteins and mucins are impor- Invitrogen). The RT-PCR product was analyzed on a 1% Ttant in many aspects of cellular metabolism and cellular Tris-acetate EDTA (TAE) agarose gel, and the expected interactions, including those involved in leukocyte trafficking (1, 1,218-bp band was excised; the DNA was then extracted from the 2). The biosynthesis of mucin-type O-glycans in animal mucins gel by using a QIAquick gel extraction kit (Qiagen). One-tenth and other glycoproteins is orchestrated by a family of N- of the product was cloned into PCR3.1(ϩ) (Invitrogen) by TA acetylgalactosaminyltransferases that transfer GalNAc to spe- cloning and sequenced. To examine the sequence of Cosmc, PCR cific Ser͞Thr residues to generate GalNAc␣1-Ser͞Thr, also was performed by using genomic DNA as the template. Because known as the Tn antigen (3). Subsequently, this precursor is the human Cosmc contains a single exon, the same primer pair was acceptor for core 1 ␤3-galactosyltransferase (C1␤3Gal-T or used for both RT-PCR and PCR of Cosmc. The PCR product T-synthase) to generate the core 1 disaccharide O-glycan Gal␤1- was analyzed on a 1% TAE agarose gel, and the expected 3GalNAc␣1-Ser͞Thr (4, 5), also known as the T antigen or 1,218-bp band was excised, purified, and directly sequenced. Thomson–Friedenrich antigen. Unlike most glycosyltrans- ferases, which occur in gene families, a single human gene on Construction of an Expression Vector Encoding C-Terminal HPC4 chromosome 7p14-p13 encodes the C1␤3Gal-T (4), although Epitope-Tagged Human C1␤3Gal-T. A mammalian expression vector there is a pseudogene for C1␤3Gal-T on chromosome 5 (un- of pcDNA4 (Invitrogen) encoding C-terminal HPC4 epitope- published data). Other core structures for mucin-type O-glycans are known, but core 1 is the common core structure found on human erythrocytes and most lymphocytes, and it serves as a This paper was submitted directly (Track II) to the PNAS office. precursor for the branched core 2 O-glycan core Gal␤1- Abbreviations: Tn antigen, GalNAc␣1-Ser͞Thr; T antigen, Gal␤1–3GalNAc␣1-Ser͞Thr; ␤ ␣ ͞ C1␤3Gal-T, core 1 UDP-Gal:GalNAc-␣-R ␤1,3-galactosyltransferase; Cosmc, core 1 ␤3-Gal- 3(GlcNAc 1-6)GalNAc 1-Ser Thr found on human leukocytes T-specific molecular chaperone; PNA, peanut agglutinin; mCosmc, mutated Cosmc, and which are required to generate ligands for P-selectin in wtCosmc, wild-type Cosmc; ER, endoplasmic reticulum. leukocyte adhesion (2, 6). The factors regulating expression of Data deposition: The sequences reported in this paper have been deposited in the GenBank core 1 O-glycans and T-synthase activity are of interest because database [accession nos. AA578739 (human EST clone for Cosmc), AC011890 (human PAC Tn and sialyl Tn antigens are recognized as tumor-associated clone RP4-655L22), and NP_067525 (mouse gene for Cosmc)]. BIOCHEMISTRY antigens for breast and colon carcinomas (7, 8), and the inability *To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.262438199 PNAS ͉ December 24, 2002 ͉ vol. 99 ͉ no. 26 ͉ 16613–16618 Downloaded by guest on September 25, 2021 tagged human C1b3Gal-T was constructed by using PCR for Cosmc on Ni-NTA Superflow and capture of C1␤3Gal-T on introducing the HPC4 epitope into the cDNA. The forward HPC4 beads, prepared as described (4), and Western blot. primer was 5Ј-GCGGATCCATGGCCTCTAAATC-3Ј. The re- verse primer containing sequence, encoding 12 aa of HPC4 Expression of Human C-Terminal HPC4 Epitope-Tagged C1␤3Gal-T and epitope (EDQVDPRLIDGK) immediately following the C- C-Terminal His-6-Tagged Cosmc and mCosmc in Hi-5 Cells. Hi-5 insect terminal proline of human C1b3Gal-T, was 5Ј-GGAAGAT- cells were cultured in EX-CELL 405 media at 27°C. For infection CTACTTGCCGTCGATCAGCCTGGGGTCCACCTGGT- or coinfection of human C1␤3Gal-T, Cosmc, and mCosmc, 0.75 CCTCAGGATTTCCTAACTTCACTTTTG-3Ј. The expected ml of baculovirus was used. At 5 days postinfection, the cells 1,144 bp of PCR product was purified on 1% TAE agarose gel were harvested, and extract was prepared for assaying and digested by NcoI and BglII. The expected 1,128-bp DNA C1␤3Gal-T activity, capture of Cosmc on Ni-NTA, C1␤3Gal-T fragment was purified and cloned into NcoI (partially digested)͞ capture on HPC4 beads, and Western blot. BamHI sites of pcDNA4 and its sequence was confirmed. For construction of an insect cell expression vector, the PCR product Transfection of Jurkat Cell with Human HPC4 Epitope-Tagged was digested with BamHI and BglII, and the 1,134-bp DNA C1␤3Gal-T and Cosmc. Jurkat cells were transiently transfected with fragment was purified and cloned into the BamHI site of expression vectors encoding a human HPC4 epitope-tagged ␤ ͞ pVL1393 (PharMingen). Thus, a baculovirus transfer vector C1 3Gal-T and or the expression vector encoding human Cosmc encoding a C-terminal HPC4 epitope-tagged human C1b3Gal-T by using GENEPORTER transfection reagent and BOOSTER (Gene in pVL1393 was constructed. Therapy Systems, San Diego). Cells were harvested at 72 h post- transfection, and a cell extract was prepared. Using these transfec- Construction of an Expression Vector Encoding Human C-Terminal tion protocols, we also identified and established stably transfected His-6-Tagged Cosmc. A cDNA encoding Cosmc with a C-terminal Jurkat cells expressing human wtCosmc. These cells were tested for His-6 tag was generated by introducing the His-6 tag into Comsc the ability of wtCosmc to complement the expression of core 1 cDNA by using PCR and EST AA578739 (Genome Systems) as O-glycans. Either mock-transfected Jurkat cells or Jurkat cells ϩ stably transfecting wtCosmc (3–5 ϫ 105 cells) were washed with a template.
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