Abstract
D2 is a membrane glycoprotein that is believed to function as a cell adhesion molecule (CAM) in neural cells. We have examined its biosynthesis in cultured fetal rat brain neurones. We found D2-CAM to be synthesized initially as two polypeptides: Mr 186,000 (A) and Mr 136,000 (B). With increasing chase times the Mr of both molecules increased to 187,000-201,000 (A) and 137,000-158,000 (B). These were similar to the sizes of D2-CAM labeled with [14C]glucosamine, [3H]fucose and [14C]mannosamine, indicating that the higher Mr species are glycoproteins. In the presence of tunicamycin, which specifically blocks the synthesis of high mannose cores, Mr were reduced to 175,000 (A) and 124,000 (B). Newly synthesized A and B are susceptible to degradation by endo-beta-N-acetyl-glucosaminidase H, which specifically degrades high mannose cores, but they are resistant to such degradation after 150 min of posttranslational processing. Hence, we deduce that A and B are initially synthesized with four to five high mannose cores which are later converted into N-linked complex oligosaccharides attached to asparagine residues. However, no shift of [35S]methionine radioactivity between A and B was detected with different pulse or chase times, showing that these molecules are not interconverted. Thus, our data indicate that the neuronal D2-CAM glycoproteins are derived from two mRNAs.
Highlights
D2 is a membrane glycoprotein that is believed to function as a cell adhesion molecule (CAM) in neural cells
'Abbreviations used in this paper: CAM, cell adhesion molecule ; DME, Dulbecco's modified Eagle's medium ; Endo H, endo-fl-acetylglycosaminidase H; GFAP, glial fibrillary acidic protein; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride ; SSF, serum-substituting factors
Within 20 to 50 min these are converted to more heterogeneous groups of larger molecules of Mr 187,000-201,000 (A) and 137,000-157,000 (B) which correspond to the sizes of two glycosylated molecules detected after longer labeling periods
Summary
Materials : Dulbecco's modified Eagle's medium (DME), methioninefree DME, basal minimal essential medium and mycoplasma-free horse serum were obtained from Gibco Laboratories (Grand Island, NY). Tissue Culture : Tissue culture flasks (4 x 6.5 cm) were pretreated with poly-L-lysine (10 jg/ml Hz0, 5 ml/flask) for 4-6 h at room temperature They were rinsed twice with phosphate-buffered saline (PBS), incubated overnight at 37°C with DME containing 20% horse serum. Further culture was continued in 5 ml serum-free media that contained serum-substituting factors (SSF) as described by Bottenstein et al (6) : transferrin (5 jug/ml), progesterone (2 x 10-8 M), putrescine (100 uM), insulin (5 ug/ml), and selenium (3 x 10 -8 M). This culture system yields nearly pure neuronal cultures after 6 d in vitro, as determined in our laboratory and by others (6-8). Endo-H (6 mU/100 ul) for 5 h at 37°C in acetate buffer (0 .05 M, pH 5.0) with CaCIZ (0.09 M) and 0.2% SDS
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