A unique molecular chaperone Cosmc required for activity of the mammalian core 1 beta 3-galactosyltransferase

Abstract

Human core 1 beta3-galactosyltransferase (C1beta3Gal-T) generates the core 1 O-glycan Galbeta1-3GalNAcalpha1-SerThr (T antigen), which is a precursor for many extended O-glycans in animal glycoproteins. We report here that C1beta3Gal-T activity requires expression of a molecular chaperone designated Cosmc (core 1 beta3-Gal-T-specific molecular chaperone). The human Cosmc gene is X-linked (Xq23), and its cDNA predicts a 318-aa transmembrane protein ( approximately 36.4 kDa) with type II membrane topology. The human lymphoblastoid T cell line Jurkat, which lacks C1beta3Gal-T activity and expresses the Tn antigen GalNAcalpha1-SerThr, contains a normal gene and mRNA encoding C1beta3Gal-T, but contains a mutated Cosmc with a deletion introducing a premature stop codon. Expression of Cosmc cDNA in Jurkat cells restored C1beta3Gal-T activity and T antigen expression. Without Cosmc, the C1beta3Gal-T is targeted to proteasomes. Expression of active C1beta3Gal-T in Hi-5 insect cells requires coexpression of Cosmc. Overexpression of active C1beta3Gal-T in mammalian cell lines also requires coexpression of Cosmc, indicating that endogenous Cosmc may be limiting. A small portion of C1beta3Gal-T copurifies with Cosmc from cell extracts, demonstrating physical association of the proteins. These results indicate that Cosmc acts as a specific molecular chaperone in assisting the foldingstability of C1beta3Gal-T. The identification of Cosmc, a uniquely specific molecular chaperone required for a glycosyltransferase expression in mammalian cells, may shed light on the molecular basis of acquired human diseases involving altered O-glycosylation, such as IgA nephropathy, Tn syndrome, Henoch-Schönlein purpura, and malignant transformation, all of which are associated with a deficiency of C1beta3Gal-T activity.

Fig 1.

Human Cosmc cDNA and deduced protein sequence and mutation of Cosmc in human Jurkat cells. (A) The nucleotide and deduced amino acid sequences of human Cosmc are shown. The cDNA predicts a 318-aa protein with a type II topology. The putative transmembrane domain is double underlined. The portions of the sequence that correspond to the identified N-terminal sequence copurified with the purified rat liver C1β3Gal-T (5) are indicated by the single underlining. The potential N-glycosylation site is boxed. The asterisk denotes the position of the T deletion described below. (B) The cDNA sequence of Cosmc was obtained by RT-PCR using total RNA from Jurkat, Molt-4, and K562 cells. The arrow indicates that portion of the sequence with a T deletion at base pair 478. (C) The T-deletion mutation at base pair 478 in Cosmc from Jurkat cells is indicated by the red box causing a truncation and introducing a stop codon. (D) Diagram of the domain organization of wtCosmc and mCosmc from Jurkat cells.

Fig 2.

Requirement of wtCosmc, but not mCosmc, for the activity of human C1β3Gal-T. (A and D) The human C-terminal HPC4 epitope-tagged C1β3Gal-T was expressed in Hi-5 insect cells with a baculovirus vector (A) or human 293T cells by transient transfection (D) with or without the coexpression of wtCosmc and mCosmc, as indicated. Infected Hi-5 cells were harvested 5 days postinfection. Extracts of the cells were prepared, and total activity of C1β3Gal-T was determined. (B and E) Extracts were incubated with Ni-NTA Superflow, and the total activity of bound C1β3Gal-T was determined. (C and F) Extracts were incubated with HPC4 beads, and the total activity of bound C1β3Gal-T was determined. The cell extracts from the above transfections of both Hi-5 cells (G) or human 293T cells (H) were analyzed by SDS/PAGE and Western blot with mAb to the HPC4 epitope present at the C terminus of the recombinant C1β3Gal-T. Lane 1 represents total cell extracts; lane 2 represents the material not bound by Ni-NTA; lane 3 represents material bound by Ni-NTA. Molecular weight markers are indicated. The sets of lanes 1–3 from each of the cell extracts derived from different transfections (or from mock-transfected cells) from A–F are indicated.

Fig 3.

Complementation of mCosmc in Jurkat cell with wtCosmc. (A) Jurkat cells were transiently transfected with expression vectors encoding the full-length human C-terminal HPC4 epitope-tagged C1β3Gal-T and/or the expression vector encoding human wtCosmc. At 72 h posttransfection, cell homogenates were prepared, and a portion was removed for assaying activity of C1β3Gal-T. (B) A portion of the extracts was incubated with HPC4 beads, and the total activity of bound C1β3Gal-T was determined. (C) Jurkat cells were stably transfected with a soluble, N-terminal HPC4 epitope-tagged form of C1β3Gal-T. The media from the cells were removed and incubated with HPC4 beads, and the total activity of bound C1β3Gal-T was determined. (D) Jurkat cells were either mock-transfected or stably transfected with cDNA encoding wtCosmc. Some cells were treated with A. ureafaciens neuraminidase. Both treated and untreated cells were incubated with fluorescently labeled Alexa488-PNA. The phase-contrast images of the cells were then monitored by phase contrast and fluorescence microscopy. (E) Jurkat cells stably expressing the soluble, N-terminal HPC4 epitope-tagged form of C1β3Gal-T (4) were incubated with or without lactacystin (10 μM) for 12 h. Cell extracts were then prepared and then separated by SDS/PAGE, and the level of the HPC4 epitope-tagged C1β3Gal-T was examined by Western blot with the HPC4 mAb.

Fig 4.

Model of interactions between Cosmc and C1β3Gal-T in generation of active enzyme. Cosmc (C) is predicted to have a chaperone function in associating with inactive C1β3Gal-T (U) in complexes that may also contain an active form of the enzyme (A). Potential associations of oligomeric complexes are indicated as CUA and CUUC, which may copurify as shown in Fig. 2. After potential rounds of binding and dissociation between Cosmc and enzyme, stable active forms of the enzyme, either dimeric (AA) or monomeric (A), are generated. Other potential chaperones not yet defined may also be involved in formation of active enzyme.