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And 0-Linked Oligosaccharides of the HLA-DR-Associated Invariant Chain

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And 0-Linked Oligosaccharides of the HLA-DR-Associated Invariant Chain Proc. Nati. Acad. Sci. USA Vol. 81, pp. 1287-1291, March 1984 Biochemistry Monensin prevents terminal glycosylation of the N- and 0-linked oligosaccharides of the HLA-DR-associated invariant chain and inhibits its dissociation from the a-,8 chain complex (carboxylic ionophores/class II andgens/post-translational processing) CAROLYN E. MACHAMER AND PETER CRESSWELL Department of Microbiology and Immunology, Duke University Medical Center, Durham, NC 27710 Communicated by D. Bernard Amos, October 24, 1983 ABSTRACT In B-lymphoblastoid cells, the HLA-DR-as- endocytosis is most likely a result of the increased pH inside sociated invariant chain is processed to a form containing 0- prelysosomal endosomes observed in monensin-treated cells linked as well as N-linked oligosaccharides. After neuraminidase (15). treatment, the O-linked carbohydrate is susceptible to diges- We previously described post-translational modifications tion with an endoglycosidase (endo-fi-N-acetylgalactosamini- of the N-linked glycan units of the invariant chain and sug- dase) that cleaves glycans with the structure Gal(l-+3)- gested that 0-linked carbohydrate was-added during its tran- GaINAc-Ser/Thr, and sialic acid can be added back to this sient association with HLA-DR antigens (5). Here we prove core oligosaccharide by specific sialyltransferases. Treatment the addition of 0-linked glycans and report the effects of of cells with the sodium ionophore monensin markedly affects monensin on the post-translational processing of the invari- the post-translational processing of the invariant chain, al- ant chain and on its interaction with HLA-DR antigens in B- though that of associated a and ,3 chains is minimally affected. lymphoblastoid cells. Only a small portion of the N-linked carbohydrate on the in- variant chain is processed to an endoglycosidase-H-resistant MATERIALS AND METHODS form. The sialic acid residues normally found on the O-linked Cells, Radiolabeling, Immunoprecipitation, and Electro- glycans are not added, but at least the first residue, GaINAc, is phoresis. Growth and radiolabeling of the Swei cell line with added. In addition to the changes in glycosylation, an intracel- [35S]methionine, immunoprecipitation of HLA-DR antigens lular accumulation of HLA-DR antigens also occurs in monen- with serum 247/HSB, and two-dimensional polyacrylamide sin-treated cells. The accumulation of HLA-DR antigens and gel electrophoresis (2D-PAGE) utilizing nonequilibrium pH the overall slower turnover rates of the a, .3, and invariant gradient separation in the first dimension were all performed polypeptides observed after monensin treatment probably re- as described (5). For experiments in which cells were cul- flects the build-up of newly synthesized proteins in Golgi appa- tured in the presence of monensin (Calbiochem), a 10 mM ratus-derived vacuoles coupled with a decrease in normal deg- stock solution in ethanol was diluted into culture medium radation in lysosomes. immediately before use. Glycoprotein Preparation. Lens culinaris hemagglutinin- At the cell surface, class II histocompatibility antigens con- binding glycoproteins were prepared as described (5). Glyco- sist of two noncovalently associated transmembrane glyco- proteins binding Bandeireae simpliciolia isolectin I (Pierce) proteins, a and p, encoded within the major histocompatibil- were isolated in a similar fashion on Bio-Gel A-15m beads ity complex. They are expressed predominantly on B cells conjugated with the lectin at 1 mg/ml. Glycoproteins were and monocytes (reviewed in ref. 1). The HLA-DR antigens eluted in 0.5 M galactose, dialyzed, and recovered by ace- are the best-characterized human class II antigens. tone precipitation. The HLA-DR antigens associate intracellularly with a Enzyme Treatments. Neuraminidase digestions were per- third polypeptide called the invariant (I) chain (2-5). A simi- formed on immunoprecipitates with 0.02 international units lar polypeptide has been observed to associate with murine of Vibrio cholerae enzyme (Calbiochem) (5). For digestion Ia antigens (6, 7). The I chain is glycosylated and methio- with endoglycosidase H, immunoprecipitates were first elut- nine-rich, has a basic isoelectric point (3, 5, 8), and is not ed in 50 ,ud of 1% NaDodSO4 at 100'C for 3 min, diluted to 0.5 encoded in the major histocompatibility complex (9, 10). Its ml with citrate/phosphate-buffered saline, pH 6.5, and incu- apparent intracellular and transient association with a and p bated with 15 milliunits of endo-/-N-acetylglucosaminidase chains suggests it may function in facilitating a-a, associa- H from Streptococcus plicatus (endo-H, Miles) for 16 hr at tion, intracellular transport of the complex, or both (4, 8). 370C. Samples were recovered by acetone precipitation. The carboxylic ionophore monensin catalyzes the ex- Endo-f3-N-acetylgalactosaminidase (endo-GalNAcase) change of Na+ and K+ across biological membranes (11). It purified from Streptococcus pneumoniae (16) was the gener- inhibits intracellular transport ofnewly synthesized polypep- ous gift of T. Beyer. Immunoprecipitates were treated with tides to the cell surface and endocytosis of ligands and re- neuraminidase, washed, and incubated in citrate/phosphate- ceptors entering the cell (reviewed in ref. 12). In monensin- buffered saline, pH 6.5, with 30 milliunits of endo-GalNAcase treated cells newly synthesized proteins accumulate in intra- for 16 hr at 370C. A control precipitate was incubated with- cellular vacuoles that appear to be derived from the Golgi out enzyme. In some experiments, the immunoprecipitates apparatus, and terminal glycosylation is incomplete in many were denatured with NaDodSO4 before digestion with endo- cases. Monensin may define a subcompartment between the GalNAcase. functionally defined cis and trans Golgi apparatus, recently For treatment with sialyltransferases, 247/HSB precipi- termed medial (13, 14). The inhibitory effect of monensin on Abbreviations: endo-GalNAcase, endo-3-N-acetylgalactosaminidase; The publication costs of this article were defrayed in part by page charge endo-H, endo-,B-N-acetylglucosaminidase H; I chain, invariant payment. This article must therefore be hereby marked "advertisement" chain; 2D-PAGE, two-dimensional polyacrylamide gel electropho- in accordance with 18 U.S.C. §1734 solely to indicate this fact. resis. 1287 1288 Biochemistry: Machamer and Cresswell Proc. NatL Acad Sci. USA 81 (1984) tates were first digested with neuraminidase. After several A 13 Ip washes, pellets were resuspended in 75 1.l of 0.1 M sodium I *-Ip 0: cacodylate buffer, pH 6.5, containing bovine serum albumin a #-I at 1 mg/ml, Triton X-100 at 10 mg/ml, and 12 mM CMP-N- acetylneuraminic acid (substrate). Approximately 10-15 mil- liunits of both f3-galactoside (a2-*3)sialyltransferase and a- C D N-acetylgalactosaminide (a2--6)sialyltransferase were add- ed. These enzymes were purified from porcine submaxillary 0- 0- gland (17, 18) and were given by A. Eckhardt and R. Mullin. A control precipitate was incubated in buffer containing sub- strate only. After 8 hr at 370C, another aliquot of each en- zyme and 5 nmol more substrate were added, and incubation E .....F was continued for a further 12 hr. I I I a Binding Assay and Solid-Phase Radioimmunoassay. HLA- 164 DR antigens on the cell surface or in total cell extracts were .4 quantitated with the monoclonal antibody XD5.A11, which recognizes a monomorphic determinant on class II P chains (unpublished data). Surface antigen expression was deter- mined with a cellular binding assay using Swei cells per- FIG. 1. The I chain possesses 0-linked carbohydrate. Swei cells formed as described (19). were labeled with [35S]methionine for 4 hr, and HLA-DR antigens Total cellular HLA-DR antigens were quantitated with a were immunoprecipitated from detergent extracts with serum and 2D-PAGE. was from solid-phase competitive radioimmunoassay. Cells were ex- 247/HSB analyzed by Electrophoresis right to left (acidic to basic) in the first dimension and from top to tracted in 1% Triton X-100 at5 x 106 cells per ml as de- bottom in 10.5% polyacrylamide/NaDodSO4 gels for the second di- scribed (5). Dilutions of the extracts in 10 mM Tris HCl, pH mension. a, 13, and I chains are indicated. Ip is the I chain processed 8.0/0.15 M NaCl/0.5% deoxycholate/1% bovine serum to a form containing sialylated N- and 0-linked oligosaccharides. albumin were prepared and a standardized amount of The arrowhead marks an immunoglobulin light chain present in the XD5.A11 antibody was added to each tube. While this incu- immunoprecipitates, which serves as a convenient marker on the bation continued for 60 min, 50ul of Lens culinaris hemag- gels. The precipitates were treated in the following manner: (A) un- glutinin-binding B-cell line membrane glycoproteins (50 treated control; (B) neuraminidase-treated; (C) neuraminidase- and ,g/ml in 5.5 M urea) were adsorbed to the wells of flexible endo-GalNAcase-treated; (D) neuraminidase-treated and denatured microtiter plates (Dynatech, Alexandria, VA). The in NaDodSO4 followed by endo-GalNAcase digestion; (E) neur- glycopro- aminidase-treated and incubated with substrate for sialyltransfer- tein solution was removed, and the plate was incubated in 10 ases only; (F) neuraminidase-treated and incubated with substrate mM Tris'HCl, pH 8.0/0.15 M NaCl/1% bovine serum albu- plus a-N-acetylgalactosaminidase (a2- 6)sialyltransferase and f-ga- min for 10 min, followed by three washes with deionized wa- lactoside (a2-*3)sialyltransferase.
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