Re-expression of nonglycosylated surface IgA in trypsin-treated MOPC 315 plasmacytoma cells.

The importance of glycosylation for the re-expression of surface immunoglobulin in trypsin-treated MOPC 315 plasmacytoma cells was examined by using tunicamycin, an antibiotic that prevents glycosylation by inhibiting the formation of N-acetylglucosamine-lipid intermediates. Tunicamycin greatly inhibited the secretion of nonglycosylated MOPC 315 IgA in trypsin-treated cells. Two hours after trypsin treatment, there was an 80% inhibition of secretion as measured by immunoprecipitation assays of biosynthetically labeled immunoglobulin. However, tunicamycin had no effect on the time course of re-expression of surface IgA in these cells as measured by TNP-sheep erythrocyte rosette formation and [125I] TNP-albumin binding to the plasmacytoma cells. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 125I-labeled cell surface IgA re-expressed in the presence of tunicamycin revealed a protein with an apparent m.w. identical to nonglycosylated MOPC 315 alpha-chains, further suggesting that nonglycosylated surface IgA was being inserted into the plasma membrane. This protein did not bind to concanavalin A-Sepharose. These data suggest that in MOPC 315 plasmacytoma cells, glycosylation is necessary for immunoglobulin secretion but not for immunoglobulin expression at the cell surface.     

Antibody-mediated targeting of liposomes. Binding to lymphocytes does not ensure incorporation of vesicle contents into the cells

Small sonicated lipid vesicles containing the water-souble fluorescent dye 6-carboxyfluorescein were formed from dioleoyl phosphatidylcholine and the antigenic lipid N-dinitrophenylaminocaproyl phosphatidylethanolamine. When these vesicles were incubated with trinitrophenyl-modified human lymphocytes and divalent anti-trinitrophenyl antibody, the antibody bound 5000 to 15 000 vesicles to each cell. Binding was detected by fluorescence microscopy and quantitated by fluorometry and flow microfluorometry. Binding was three times greater with F(ab')2 fragments than with the whole antibody and, as expected, was almost absent with the monovalent F(ab') fragments. It was also absent or greatly reduced, (i) with control immunoglobulin G, (ii) in the presence of excess soluble trintrophenyl hapten, or (iii) if hapten was omitted from either cells or vesicles. It was unaffected by sodium azide and 2-deoxy-D-glucose but was markedly decreased at 3 degrees C. It was not reversed by incubation at 3 degrees C with excess trinitrophenyl lysine. Self-quenching of the fluorescence of 6-carboxyfluorescein was used to distinguish between release of vesicle contents into the cells and simple binding of intact vesicles (Weinstein, J.N., Yoshikami, S., Henkart, P., Blumenthal, R. and Gagins, W.A. (1977) Science 195, 489--491). Antibody-mediated binding led to little or no increase over spontaneous background levels in the amount of vesicle contents released into the lymphocytes.     

Resonance Raman-spectroscopic studies of the hapten features involved in the binding of 2,4-dinitrophenyl haptens by the mouse myeloma proteins MOPC 315 and MOPC 460.

The binding of four dinitrophenyl haptens to the mouse myeloma proteins MOPC 315 IgA (immunoglobulin A) and MOPC 460IgA was studied by resonance Raman spectroscopy. Isotopic substitution with 15N and 2H was used to assign features in the resonance Raman spectra of the free haptens. Changes in each of these features on binding to the proteins could then be attributed to interactions of the proteins' binding sites with either the p-NO2 or the o-NO2/amine regions of the haptens. The interactions between a given hapten and MOPC 315 IgA are often quite distinct from those between the same hapten and MOPC 460 IgA. Moreover, for both antibodies the nature of the R side chain in a Dnp-NHR (Dnp, 2,4-dinitrophenyl) compound appears to modify the interactions between the Dnp chromophore and the protein. Thus, with the haptens studied, there is no unique set of contacts between the Dnp group and the binding site. The contacts expected between epsilon-2,4-dinitrophenyl-L-lysine and the site on MOPC 315 IgA, on the basis of a recent model for this site [Dwek, Wain-Hobson, Dower, Gettins, Sutton, Perkins & Givol (1977) Nature (London) 266, 31--37] were not detected. However, the contacts between this hapten and the site on MOPC 460 IgA were closer to those predicted by the model for MOPC 315 IgA.     

Comparison of the dimensions of the combining sites of the dinitrophenyl-binding immunoglobulin A myeloma proteins MOPC 315, MOPC 460 and XRPC 25 by spin-label mapping

The mouse immunoglobulin A myeloma proteins MOPC 315, MOPC 460 and XRPC 25 all possess dinitrophenyl (Dnp)-binding activity. Differences in specificities were shown by measuring the affinities of a variety of haptens. By using a series of Dnp-spin-labelled haptens, the dimensions of the binding sites of the three myeloma proteins were compared by the method described for protein MOPC 315 [Sutton, Gettins, Givol, Marsh, Wain-Hobson, Willan & Dwek (1977) Biochem. J.165, 177-197]. The dinitrophenyl ring is rigidly held in all three sites. The depths of the sites are all 1.1-1.2nm, but there are differences in the lateral dimensions at the entrance to the sites. For protein XRPC 25 these dimensions are 0.75nmx0.8nm, which may be compared with 0.85nmx1.1nm for protein MOPC 315 and >/=1.0nmx1.1nm for protein MOPC 460. The site in protein MOPC 460 is more symmetrical with respect to the plane of the dinitrophenyl ring than in either of the other two myeloma proteins and also allows greater penetration of solvent. In protein XRPC 25 a positively charged residue was located at the entrance to the site, similarly positioned to that reported for protein MOPC 315 [Sutton, Gettins, Givol, Marsh, Wain-Hobson, Willan & Dwek (1977) Biochem.J.165, 177-197]. All three proteins possess lanthanide-binding sites, but only in protein MOPC 315 is there antagonism between lanthanide and hapten binding. However, the effects of the diamagnetic La(III) on the electron-spin-resonance spectra of bound Dnp spin labels in both proteins MOPC 460 and XRPC 25 suggest an interaction between the two sites. Comparison of this effect with that caused by the addition of the paramagnetic Gd(III) enables the distance between the lanthanide- and hapten-binding sites to be calculated. In both proteins MOPC 460 and MOPC 315 the metal site is approx. 1.0nm from the nitroxide moiety of the spin-labelled hapten, but in protein XRPC 25 this distance is at least 2.0nm.     

Small-angle neutron scattering studies of the conformation of myeloma protein MOPC315 and its Fab fragment, and the interaction with a monovalent dinitrophenyl hapten

The first small-angle scattering study of an immunoglobulin A is reported. Neutron measurements have been made to determine conformational parameters of the mouse myeloma protein MOPC315 and to relate these to previous immunoglobulin G results. Use of the contrast method shows that the MOPC315 IgA molecule is not simply globular, that it has a dry volume of 220.0 +/- 4.5 nm3 corresponding to a mass density of 1.275 +/- 0.025 g cm-3 and that its full and cross-sectional radii of gyration, corrected for concentration dependence, are 7.97 +/- 0.07 nm, 2.40 +/- 0.08 nm and 1.33 +/- 0.07 nm respectively. Similar study of its Fab fragment gives a dry molecular volume of 69.0 +/- 0.7 nm3, a mass density of 1.285 +/- 0.015 g cm-3 and uncorrected radii of gyration that are consistent with those of the parent and support an overall "T" or "Y" conformation in solution. Addition to saturation of a small monovalent dinitrophenyl hapten leaves the dry volume of the whole molecule unaltered, but may slightly lower one or more of its radii of gyration. The significance of this finding is discussed. Comparative studies with rabbit anti-dinitrophenyl immunoglobulin G antibody suggest a different initial conformation but similar consequences of hapten binding, which, if real, are probably unrelated to classical complement fixation.     

The role of nitro groups in the binding of nitroaromatics to protein MOPC 315.

Two series of dinitrophenyl haptens, in which chlorine replaces one or both nitro groups, were used to investigate, by a combination of high-resolution 1H n.m.r. and fluorescence quenching, the presence of groups in the combining site of protein MOPC 315, which form hydrogen bonds to the aromatic-ring substituents of the hapten. The large differences in binding constants on successive replacement of nitro groups were shown to be due to specific hapten-substituent-protein interactions by (a) showing that there was little difference in the interaction between these haptens and 3-methylindole (a model for the residue tryptophan-93L with which the hapten stacks in protein MOPC 315), (b) proving by 1H n.m.r. that the mode of hapten binding is constant and (c) showing that the differences in Kd were consistent with the relative hydrogen-bonding capacities of chlorine and the nitro moiety. In this way it was established that each nitro group forms a hydrogen bond. Furthermore, from consideration of the 1H n.m.r. chemical shifts of several dinitrophenyl haptens and their trinitrophenyl analogues, it was shown that there is no distortion of the o-nitro group on binding to the variable fragment of protein MOPC 315.     

Disappearance of macrophage surface folds after antibody-dependent phagocytosis

We have employed the method of Burwen and Satir (J. Cell Biol., 1977, 74:690) to measure the disappearance of surface folds from resident guinea pig peritoneal macrophages after antibody-dependent phagocytosis. Unilamellar phospholipid vesicles containing dimyristoylphosphatidylcholine and 1 mol % dinitrophenyl-epsilon- aminocaproyl-phosphatidylethanolamine, a lipid that possesses a hapten headgroup, were prepared by an ether injection technique. These vesicles were taken up by macrophages in a time- and temperature- dependent fashion. Vesicles that contained ferritin trapped in the internal aqueous volume were identified within macrophages by transmission electron microscopy. Scanning electron microscopy has shown that macrophage surface folds decrease dramatically after phagocytosis. The surface fold length (micrometer) per unit smooth sphere surface area (micrometer2) decreases from 1.3 +/- 0.3 micrometer- 1 to 0.53 +/- 0.25 micrometer-1 when cells are incubated in the presence of specific anti-DNP antibody and vesicles at 37 degrees C. No significant effect was observed in the presence of antibody only or vesicles only. Our studies shown that phagocytosis is associated with a loss of cell surface folds and a loss of cell surface area, which is consonant with current views of the endocytic process. On the basis of our uptake data, we estimate that approximately 400 micrometer2 of vesicle surface membrane is internalized. The guinea pig macrophage plasma membrane has a total area of approximately 400 micrometer2 in control studies, whereas the cells have roughly 300 micrometer2 after phagocytosis. These estimates of surface areas include membrane ruffles and changes directly related to changes in cell volume. We suggest that during antibody-dependent phagocytosis a membrane reservoir is made available to the cell surface.     

Inhibition of interleukin-10 (IL-10) production from MOPC 315 tumor cells by IL-10 antisense oligodeoxynucleotides enhances cell-mediated immune responses

 Interleukin-10 (IL-10) has both inhibitory and stimulatory effects on diverse cell types of the immune system. It inhibits the antigen-presenting capacity of monocytes/macrophages and stimulates T cell proliferation. Although many tumors spontaneously release IL-10, the physiological relevance of this phenomenon to the in vivo antitumor immune response is not known. To elucidate the physiological role of tumor-released IL-10, we used IL-10-specific antisense oligodeoxynucleotides (AS-ODN) for the inhibition of IL-10 production from the tumor cells. Incubation of MOPC 315 plasmacytoma with IL-10 AS-ODN in vitro resulted in inhibition of IL-10 production and also in enhancement of the expression of major histocompatibility complex (MHC) class I, MHC class II, and B7-1 molecules. MOPC 315 cells incubated with IL-10 AS-ODN (MOPC-IL10AS) for 16 h in vitro showed reduced tumorigenicity in Balb/c mice. The mice implanted with MOPC-IL10AS effectively rejected the tumor graft, and showed strong cytotoxic T lymphocyte (CTL) activity against the parental MOPC 315 cells. In addition, MOPC-IL10AS were more effective as stimulator cells in mixed lymphocyte/tumor cell culture, and as target cells in a CTL assay. These results imply that IL-10 spontaneously released from MOPC 315 cells inhibits their immunogenicity and that the inhibition of IL-10 production by IL-10 AS-ODN may be a way to enhance the host cellular antitumor immune response. Received: 11 November 1999 / Accepted: 6 April 2000     

Studies of the mechanism of tunicamycin in hibition of IgA and IgE secretion by plasma cells.

Tunicamycin, an antibiotic which blocks the formation of N-acetylglucosamine-lipid intermediates, thereby preventing glycosylation of glycoproteins, inhibits the secretion of IgA and IgE by MOPC 315 mouse plasma cells and IR162 rat plasma cells, respectively. At 0.5 microng of tunicamycin per ml, D-[14C]glucosamine incorporation into newly synthesized immunoglobulin was inhibited greater than 90% while the overall rate of protein synthesized was much less inhibited (40% in the case of MOPC 315 cells and 13% in the case of IR162 cells). This dose of tunicamycin produced an 85% inhibition of IgA secretion by the MOPC 315 cells and a complete inhibition of intact IgE secretion by the IR162 plasma cells. In contrast, tunicamycin had little effect on the secretion of normally nonglycosylated lambda light chains or on cell-free protein synthesis, demonstrating that tunicamycin is not a general inhibitor of protein synthesis or a non-specific inhibitor of protein secretion. No enhancement of intracellular degradation of nonglycosylated immunoglobulin could be demonstrated. Electron microscopy of tunicamycin-treated MOPC 315 cells revealed marked dilatations of the rough endoplasmic reticulum, and direct immunofluorescence indicated that the dilated rought endoplasmic reticulum contained IgA. These data indicate that glycosylation of newly synthesized IgA and IgE may be necessary for normal secretion to occur.     

The binding of 2,4,6-trinitrophenyl derivatives to the mouse myeloma immunoglobulin A protein MOPC 315

The binding of Tnp (2,4,6-trinitrophenyl) derivatives to the Fv fragment (variable region of heavy and light chains) of the mouse myeloma IgA protein MOPC 315 was investigated by 270MHz proton nuclear magnetic resonance. Two of the haptens, Tnp-glycine and Tnp-l-aspartate, are in fast exchange with the Fv fragment, and the changes in chemical shifts for both protein and hapten resonances were determined by titrations. For the tightly binding hapten epsilon-N-Tnp-alpha-N-acetyl-l-lysine, which is in slow exchange with the Fv fragment, the changes in chemical shifts for the hapten H(3)+H(5) resonances were determined by cross-saturation. By using these data and the known structure of the combining site of protein MOPC 315 [Dwek, Wain-Hobson, Dower, Gettins, Sutton, Perkins & Givol (1977), Nature (London) 266, 31-37] the mode of binding of Tnp derivatives is deduced by ring-current calculations. The trinitrophenyl ring stacks with tryptophan-93(L) (light chain) in the ;aromatic box' formed by tryptophan-93(L), tyrosine-34(L) and phenyl-alanine-34(H) (heavy chain). Further evidence for the stacking interaction with a tryptophan residue is provided by the similarity of the optical-difference spectra observed with Tnp-aminomethylphosphonate in the presence of either the Fab fragment (light chain and N-terminal half of heavy chain) of protein MOPC 315 or tryptophan. These data show that the modes of binding of all the Tnp derivatives are very similar, despite a 100-fold range in their affinities. It is also concluded that the modes of binding of Dnp (2,4-dinitrophenyl) and Tnp derivatives to protein MOPC 315 are very similar, and that the structural basis for this is that the aromatic box is large enought to allow the trinitrophenyl ring to stack with tryptophan-93(L) while still forming hydrogen bonds to asparagine-36(L) and tyrosine-34(L).     

Biosynthesis of immunoglobulin A (IgA) and immunoglobulin M (IgM). Requirement for J chain and a disulphide-exchanging enzyme for polymerization

Mouse myeloma cells secreting 19S IgM (immunoglobulin M) (MOPC 104E and TEPC 183) or monomer and polymer IgA (immunoglobulin A) (MOPC 315) were incubated with radioactive leucine and the intracellular and secreted immunoglobulins and immunoglobulin subunits were prepared by preparative sucrose-density-gradient centrifugation. Samples were reduced in the presence or absence of isolated J chain, passed over Sephadex G-25 and then incubated at 37 degrees C for 30min with or without a source of disulphide-interchange enzyme. The extent of reassembly of reduced subunits was then evaluated by electrophoresis in polyacrylamide gels. Provided that J chain and the disulphide-interchange enzyme were supplied, both IgM and IgA could be assembled from their respective subunits, obtained by reductive cleavage of polymeric forms. Under similar conditions, assembly of polymeric forms from intracellular or secreted 7S monomer subunits also occurred. Under these conditions polymerization was total, there being no residue of the monomeric form. Reassembly did not occur in the absence of either J chain or the enzyme. All of the J chain released from IgM by reductive cleavage was incorporated back into the reassembled polymer. The J chain is therefore likely to be an essential structural requirement for polymeric immunoglobulins. A variety of controls ruled out non-specific interactions, and further suggested that the amino acid sequence of polypeptide chains determines the specificity of polymerization. The fact that intracellular IgA and IgM monomer subunits known to be deficient in galactose and fucose can be completely polymerized suggests that the addition of carbohydrate does not control polymerization.     

Conformational transitions of antibodies induced by hapten binding

The conformational changes of antibody structure induced by hapten molecule binding were investigated by means of thermal perturbation difference spectroscopy. The studies of the free rabit anti-dinitrophenyl antibodies show the conformational transition at temperatures between 25 and 35 degrees C. The changes occurring at the higher temperature are accompanied by the screening of the significant part of exposed tyrosine residues. Binding of the hapten molecules induces a similar transition to that which occurs between the two temperature dependent states of the free antibody. In contrast to our previous results with anti-dansyl rabbit antibodies the dinitrophenyl lysine stabilizes the "low temperature" native state of the protein. The investigation of the MOPC-315 mouse immunoglobulin A myeloma protein possessing anti-dinitrophenyl activity indicates no conformational transition at temperatures between 25 and 35 degrees C and only a small decrease of tyrosine exposure induced by the hapten binding. Our present and previous results indicate that most of the free immunoglobulins exist in two native conformational states which have a small difference in free energy. Hapten binding causes the transition in equilibrium between the two states towards the one of better binding. It is possible that this transition is necessary but not sufficient step for inducing the effector function of antibodies.     

Magnetic resonance studies of the binding site interactions between 19F-labeled nitrophenyl haptens and specific mouse myeloma immunoglobulin MOPC-315

The interactions between MOPC-315, a mouse myeloma protein with specificity for nitrophenyl haptens, and 19F-substituted haptens have been investigated using nuclear magnetic resonance (NMR) spectroscopy. The haptens studied are mono- or dinitrophenyl derivatives of gamma-aminobutyric acid, lysine, or glycine which have trifuoromethyl groups attached to the phenyl rings. Upon binding to immunoglobulin, the 19F nucleus experiences a downfield shift whose magnitude depends on the position of the trifluoromethyl group on the phenyl ring but is independent of other structural changes in the hapten such as the number of nitro groups attached to the phenyl ring. Further, the chemical shift of bound hapten is not influenced by the amount of the constant region attached to the binding site; we accordingly conclude that the presence of the distal, constant regions of the immunoglobulin molecule does not influence binding site interactions.     

The murine IgA-secreting plasmacytoma MOPC-315 expresses somatostatin receptors

We have previously shown that some neuropeptides had a profound effect on in vitro Ig synthesis (especially IgA) and mitogen-driven murine lymphocyte proliferation. MOPC-315, an IgA-secreting plasmacytoma line, has been extensively used in studies of the regulation of IgA synthesis. In this report we show that the neuropeptide somatostatin (SOM) inhibits proliferation ([3H]thymidine uptake) of MOPC-315 and also inhibits IgA synthesis in vitro. MOPC-315 cells bind both fluorescent SOM and [125I]SOM specifically. On cytofluorimetric analysis, 68 +/- 6.8% (mean +/- SE, n = 7) of MOPC 315 cells labeled with fluorescent SOM and this staining was compatible by incubation with an excess of unlabeled peptide. Specific [125I]SOM binding increased linearly with cell concentration, was rapid and achieved equilibrium after 20 min at 4 degrees C. It was temperature-dependent, readily reversible, and under equilibrium conditions demonstrated a dissociation constant of 1.6 +/- 0.7 nM (mean +/- SE, n = 5). Scatchard analysis showed that MOPC-315 cells had 40,733 +/- 16,050 (mean +/- SE) binding sites for SOM per cell. The characteristics of the interactions of SOM with MOPC-315 cells suggest a specific receptor-mediated mechanism whereby this neuropeptide may modulate lymphocyte function.     

The preendosomal compartment comprises distinct coated and noncoated endocytic vesicle populations

The transfer of molecules from the cell surface to the early endosomes is mediated by preendosomal vesicles. These vesicles, which have pinched off completely from the plasma membrane but not yet fused with endosomes, form the earliest compartment along the endocytic route. Using a new assay to distinguish between free and cell surface connected vesicle profiles, we have characterized the preedosomal compartment ultrastructurally. Our basic experimental setup was labeling of the entire cell surface at 4 degrees C with Con A-gold, warming of the cells to 37 degrees C to allow endocytosis, followed by replacing incubation medium with fixative, all within either 30 or 60 s. Then the fixed cells were incubated with anti-Con A-HRP to distinguish truly free (gold labeled) endocytic vesicles from surface-connected structures. Finally, analysis of thin (20-30 nm) serial sections and quantification of vesicle diameters were carried out. Based on this approach it is shown that the preendosomal compartment comprises both clathrin-coated and non-coated endocytic vesicles with approximately the same frequency but with distinct diameter distributions, the average noncoated vesicle being smaller (95 nm) than the average coated one (110 nm). In parallel experiments, using an anti-transferrin receptor gold-conjugate as a specific marker for clathrin-dependent endocytosis it is also shown that uncoating of coated vesicles plays only a minor role for the total frequency of noncoated vesicles. Furthermore, after perturbation of clathrin-dependent endocytosis by potassium depletion where uptake of transferrin is blocked, noncoated endocytic vesicles with Con A-gold, but not coated vesicles, exist already after 30 and 60 s. Finally, it is shown that the existence of small, free vesicles in the short-time experiments cannot be ascribed to recycling from the early endosomes.     

Possible three-dimensional backbone folding around antibody combining site of immunoglobulin MOPC167

Using a recently developed method of predicting possible three dimensional foldings of immunoglobulin backbones around antibody combining sites, we have attempted to construct the structure of immunoglobulin MOPC167 which could bind phosphorylcholine. A small pocket was present in the middle of the predicted structure similar to that of another phosphorylcholine binding immunoglobulin, McPC603, the tertiary structure of which has been determined by X-ray diffraction studies. Although detailed atomic co-ordinates of McPC603 were not available, a rough comparison between these structures strongly suggested that the foldings of complementarity determining regions were alike only for those with identical lengths and similar amino acid sequences. The predicted structure of MOPC167 as compared with the previously predicted structure of MOPC315, a 2,4-dinitrophenol binding immunoglobulin, further suggested that the light chain of one immunoglobulin could indeed combine with the heavy chain of another immunoglobulin, but might not result in a reasonable site for binding antigens.     

Immunohistochemical detection of Fc receptor. II. Electron microscopic demonstration of Fc receptor by using soluble immune complexes of ferritin-antiferritin immunoglobulin G.

The mouse mesenteric lymph node cells were incubated in the soluble immune complexes of ferritin-antiferritin immunoglobulin G at 37 degrees C for 20 min. After being washed, postfixed with OsO4 and dehydrated by degraded ethanol series, the lymph node cells were observed by electron microscope. Apprroximately 15% of the cells (mainly composed of small lymphocytes) bound ferritin particles to the cell surface. The distribution pattern of the binding of ferritin particles (ferritin-antiferritin immunoglobulin G) took the form of discrete patches of irregular distribution interspaced with unlabeled portions. The electron microscopic features of ferritin particles (ferritin-antiferritin immunoglobulin G) attached to the cell surface suggest that a structure of constant conformation (Fc receptor) situated in the cell membrane takes part in the binding of ferritin-antiferritin immunoglobulin G.     

Internalization of beta-adrenergic receptor in A431 cells involves non-coated vesicles

To characterize the mechanism of internalization of beta-adrenergic catecholamine receptors on human epidermoid A431 carcinoma cells, their distribution was analyzed by immunocytochemistry using the monoclonal anti-receptor antibody BRK2. In preconfluent cultures, the receptors appeared to be randomly distributed on the cell surface. Exposure to the agonist isoproterenol induced an overall decrease in the number of cell surface receptors as determined by binding experiments and visualized by immunofluorescence. When cells were incubated at 4 degrees C with BRK2 and anti-mouse IgG-gold and then transferred at 37 degrees C, non-coated invaginations and vesicles were labeled. The addition of isoproterenol resulted in an increased rate of internalization of the receptor-BRK2-anti-IgG-gold complex. When incubation with the two antibody reagents was prolonged (with or without isoproterenol), non-coated vesicles fused in the endosomal compartment, and receptors were transferred to multivesicular bodies and lysosomes. At no stage in this process was there any indication that clathrin-coated pits or vesicles participated. Furthermore, we found that an intracellular potassium depletion treatment known to inhibit endocytosis, did not affect the normal pattern of desensitization of beta-adrenergic receptors.     

Studies of the biosynthesis of the mannose 6-phosphate receptor in receptor-positive and -deficient cell lines

Phosphomannosyl residues present on lysosomal enzymes are specifically recognized by the mannose 6-phosphate receptor protein. This interaction results in the selective targeting of lysosomal enzymes to lysosomes. While this pathway is operative in many cell types, we have found four cultured cell lines that are deficient in the ability to bind lysosomal enzymes containing phosphomannosyl residues to their intracellular or surface membranes (Gabel, C., D. Goldberg, and S. Kornfeld, 1983, Proc. Natl. Acad. Sci. USA, 80:775-779). These cells appear to segregate lysosomal enzymes by an alternate intracellular pathway. To determine the basis for the lack of mannose 6-phosphate receptor activity in these cell lines, we studied the biosynthesis of the receptor in receptor-positive (BW5147) and receptor-deficient (P388D1 and MOPC 315) cells. The cells were labeled with [2-3H]mannose or [35S]methionine and the receptor was immunoprecipitated with an antireceptor antiserum. BW5147 cells synthesize a receptor protein whose size increases after translation/glycosylation. MOPC 315 cells produce an apparently normal receptor and degrade it rapidly. P388D1 cells fail to synthesize any detectable receptor. The receptor from BW5147 and MOPC 315 cells is a glycoprotein with both high mannose and complex asparagine-linked oligosaccharides. The complex-type units become fully sialylated and remain so during long periods of chase.     

The allogeneic effect on tumor growth. II. Suppression of both ascitic and solid MOPC 315 plasmacytoma by the graft-vs-host reaction, with pathologic correlation.

The growth of an ascitic murine plasmacytoma, MOPC 315, can be retarded in CAF1 hybrid host mice by the i.p. injection of donor lymphoid cells. The graft-vs-host reaction can be established by a variety of donor cells, including parental BALB/c and A/J and congenic inbred B10.D2 which share the major histocompatibility locus with BALB/c(H-2d). Optimal results are consistently obtained when parental BALB/c spleen cells are injected before tumor inoculation, and a second dose of donor spleen cells injected 1 week later. This aloogeneic effect on tumor growth is manifested by delayed appearance of the tumor and prolonged host survival. Pathologic studies on the ascites tumor indicated that the allogeneic effect suppresses the initial appearance and early growth of the plasmacytoma. However, once established, MOPC 315 grows rapidly and fatally in both control mice and recipients of donor lymphoid cells. Further, a subcutaneous implant of MOPC 315 is suppressed by an allogeneic effect established either i.v. with BALB/c spleen cells before tumor inoculation or by BALB/c spleen cells administered subcutaneously at the time of MOPC 315 implant. Thirty percent of mice treated by i.v. or subcutaneous donor lymphoid cells were tumor free at 150 days after tumor inoculation.