Human 8 2 - microglobulin is a Substrate

Incubation of purified human 82-microglobulin (B2-m) with tissue transglutaminase (Tgase) resulted in the formation of high molecular weight polymers revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In the presence of 30 mM ['4C]methylamine, the polymer formation was prevented, but incorporation of methylamine into ,8 2-m (equal to 1 methylamine per 1 molecule) could be observed . From the sheddings of peripheral blood mononuclear cells occurring in the presence of Tgase, it is apparent that anti-1(3 2-m immunoadsorbent removed, in addition to human leukocyte antigen (HLA) and 82-m, some other proteins . The enzyme could incorporate ['°C]methylamine into,82-m of the shedding cells. On addition of rabbit anti-human a2-m antibody, followed by fluorescenne-labeled goat anti-rabbit IgG antibody to human mononuclear blood cells, the otherwise homogeneous distribution of fluorescence turned into spots and patches on cells previously incubated with Tgase or Cat+-ionophore A23187 . MATERIALS AND METHODS Human,82-microglobulin (132-m) isolated from culture media ofhuman lymphoid cell lines was a kind gift of Dr. N. Tanigaki (Roswell Park Memorial Institute, Buffalo, New York). Guinea pig liver Tgase, which was purified according to Connellan et al. (5), exhibited 92% t 8% of the reported specific activity upon assay by hydroxamate formation with benzyloxycarbonyl (2)-r.-glutaminylglycine . Active site-inhibited Tgase prepared as previously described (15) showed no activity . An IgG fraction of monospecific rabbit anti-human #2-m-antiserum was obtained from Dako Immunoglobulins Ltd. (Copenhagen, Denmark) and diluted to 700pg/ml with Parker medium TC-199 just before use. Fluoresceinelabeled IgG of goat antiserum against rabbit IgG (Hyland Diagnostics Div., Travenol Laboratories, Inc., Costa Mesa, Calif.) was used as a second antibody at a concentration of 500 pg/ml. Ca"-ionophore A23187, a gift of Eli Lilly and Company (Indianapolis, Indiana), was dissolved in dimethytsulfoxide at l mg/ ml and stored at 4°C. ['"C]Methylamine (specific activity 40 mCi/mmol) was purchased from New England Nuclear (Boston, Massachusetts). Protein standards for molecular weight determination by sodium dodecyl sulfate (SDS) gel electrophoresis were obtained from Serva Fine Biochemicals, Inc., Garden City Park, N.Y. All other chemicals were either reagent grade or the best available. Crosslinking of 82-m by Tgase At various intervals after the addition of 3p1 ofTgase (11 mg/ml) to a 150-,a1 solution of,%-m (1 .26 mg/ml in 30 mM Tris, pH 7.5, 100 nM NaCl, 5 mM CaCl z, 2 mM DTT, l EDTA), 20-pl aliquots were withdrawn to process for SDS polyacrylamide get electrophoresis (PAGE) on disc gels (17) . When the incorporation of labeled methylamine into #2-m was studied, 30 mM [r"C]methylamine was also included in the incubation mixture . After the usual electrophoresis, staining, and destaining procedures, the band corresponding to 82-m was cut out THE JOURNAL OF CELL BIOLOGY " VOLUME 89 JUNE 81 706-710 © The Rockefeller University Press " 0021-9525/81/06/0706/05 $1 .00 on July 8, 2017 jcb.rress.org D ow nladed fom from the gel, sliced, solubilized in Soluene-350 (Packard Instrument Co., Inc., Downers Grove, Ill.), and radioactivity was measured in a Packard liquid scintillation counter type 3320 . Preparation of Human Peripheral Blood Mononuclear Cells Human peripheral blood mononuclear cells (PBMC) were separated from freshly drawnblood ofhealthy adults with a Ficoll-Uromiro (Pharmacia Uppsala, Sweden) gradient according to the method of B6yum (2) . The preparation contained 90-95% lymphocytes as determined by morphology, <3% of the cells engulfed latex particles, and 98% of the cells were viable as indicated by trypan blue exclusion. Shedding of Surface-labeled Human PBMC and Adsorption of the Shed Supernate on Sephadex Anti-#2-m Particles Supernates containing membrane components shed as a result of temperature shift (from4° to 37°C) were obtained from surface-labeled human PBMC by the method described by Sarmay et al . (l3). 0 .6 ml ofPBMC suspension (7 .5 x 107 cells/ml in serum-free Parker medium TC-199), previously surface-labeled by the lactoperoxidase technique (16) at room temperature, then washed and kept at 4°C, was incubated at 37°C for 60 min in the presence of 501ag/ml active siteinhibited Tgase, 50 wg/ml active enzyme, or 0.1 pg/ml Ca"-ionophore A23187 . After centrifugation, the protein concentration (Bio-Rad Protein Assay, Bio-Rad Laboratories, Rockville Centre, N.Y .), as well as the concentration of /32-m (Phadebas /" 2-m RIA kit, Pharmacia Fine Chemicals, Div . of Pharmacia, Inc., Piscataway, N.J .), was determined in the supernate. Then, portions of the supernates (equal to 1 .0 mg protein in each case) were mixed with 0.3-ml packed gel slurries of Sephadex anti-#2-m immunoadsorbent (3 x 10' particles/ml; Pharmacia) which is previously washed in 10 mM Tris-HCI, pH 8 .2 + 0 .1 M NaCl + 1 .5 mM EDTA . After 2 h of stirring at 37°C, the samples were further shaken at 4°C overnight . Afterwards, the slurries were centrifuged and washed free ofunbound materials with the Tris-saline buffer . Specifically bound proteins were released by being boiled for 3 min in 100 Al ofSDS-PAGE sample buffer containing 2% SDS and 2% 2-mercaptoethanol, then analyzed by SDS-PAGE . Essentially the same procedure was repeated when the Tgase-catalyzed incorporation of ["C]methylamine (final concentration : 1 mM) into the R2-m of shedding but noniodianated PBMC was studied . After electrophoresis, the gets were stained and sliced into 1-mm thick pieces ; then radioactive measurements were performed (8) . Patching of R2-m on PBMC Freshly prepared PBMC suspension was distributed into a series of plastic tubes (2 x 106 cells/tube) in a volume of200 Al of medium TC-199. In 100 Al of medium, various amounts of Tgase, active site-inhibited Tgase, Ca 2'-ionophore A23187, respectively, or medium alone, were added to the cell suspensions, which were incubated for 15 min in a waterbath at 37° C afterward. Then, the following steps were performed: (a) pelleting of the cells and washing them twice with medium TC-199 at room temperature; (b) resuspending each in a 50-p1 solution of diluted anti-human 02-antibody and incubating for 30 min at room temperature; (c) washing them twice in ice-cold medium TC-199 ; (d) resuspending each in goat anti-rabbit IgG antibody labeled with fluoresceine and incubating in an ice bath for 30 min; (e) washing them twice in ice-cold medium TC-199; and (/') fixing them in 1% paraformaldehyde diluted in phosphate-buffered saline. (In two experiments, the cells were first treated according to step (b), and only then with 5 pg of Tgase ; the following steps were the same .) After a last washing in medium TC-199, the cell pellet was resuspended in buffered glycerol and observed under a fluorescence microscope (Fluoval, Zeiss) . The proportion of cells showing spots or patches was inferred from examination of 150-200 cells/ sample . FIGURE 1 Crosslinking Of ,f32-m by tissue Tgase . Proteins were separated on 10% polyacrylamide SDS gels after incubation at 37°C and subsequent denaturation . 1 : Guinea pig liver Tgase incubated for 60 min without Ca t + . 2: Tgase incubated for 60 min in the presence of Ca t * . 3:,82-m . 4 : /92-m and Tgase incubated for 60 min without Ca" . 5-7: #2-m and Tgase incubated for 10, 30, and 60 min, respectively, in the presence of Ca21 . 8: /3 2-m and Tgase incubated for 60 min in the presence of Ca t' and 30 mM ["Clmethylamine (83,500 cpm/nmol) ; 25 .2 flg of /32-m was run on the gel ; and the cpm measured in the ,Q2-m band was 179,825 . RAPID COMMUNICATIONS 707 on July 8, 2017 jcb.rress.org D ow nladed fom