ey-globulin, labeled in its protein moiety with H3-leucine, was confined to the

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1 THE SYNTHESIS AND SECRETION OF y-globulin BY LYMPH NODE CELLS, III. THE SLOW ACQUISITION OF THE CARBOHYDRATE MOIETY OF 7-GLOBULIN AND ITS RELATIONSHIP TO SECRETION BY ROBERT M. SWENSON* AND MILTON KERN NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES, NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND Communicated by C. B. Anfinsen, December 1, 1967 The mechanism of the selective secretion of 'y-globulin by lymph node cells has been in part elucidated by biochemical studies concerned with the intracellular status of this protein. In previous reports from this laboratory,'-3 it was demonstrated that the bulk of the specifically coprecipitated intracellular ey-globulin, labeled in its protein moiety with H3-leucine, was confined to the microsome fraction of lymph node cell homogenates. Furthermore, the microsome fraction possessed a compartment in which the major part, if not all, of the H3-protein was H3-y-globulin. In contrast, when H3-glucosamine, H3- galactose, or H3-glucose was used to label the covalently linked carbohydrate group of y-globulin, less than 10 per cent of the labeled y-globulin was present in the microsome fraction. These findings unequivocally demonstrated the intracellular segregation of 7-globulin molecules into at least two classes,2 and revealed as well an intracellular selectivity for the processing of 7-globulin in comparison to nonsecreted proteins.' Although isotopic amino acids are incorporated into intracellular 7-globulin without a detectable delay, there is approximately a 20-minute lag before labeled 7-globulin appears in the extracellular medium.4 In this report, evidence is presented establishing the temporal relationship between the secretory lag and the time required for the acquisition of the carbohydrate moiety of 7-globulin. These results are discussed in regard to the mechanism of secretion of y- globulin. Materials and Methods.-The materials used and the procedures followed for the immunization of rabbits have been described previously.4 5 The techniques for the isolation and incubation of cells were as previously described, except that glucose was omitted from the cell incubation medium.", 4" It was necessary to omit glucose from the media in all our experiments because it interfered with incorporation of isotopic monosaccharides. The techniques used for the specific coprecipitation of isotopically labeled ev-globulin and the control for nonspecific coprecipitation have been described.' Precipitates were routinely harvested by centrifugation at 9000 X g for 5 min and washed twice with 0.15 NaCl-0.01 M phosphate, ph 7.0. After incubation in 3.0 ml of 5% trichloroacetic acid at 950 for 10 min, the precipitate was washed first with ethanol: ether, 3:1, then with ether, dried, and counted.6 In all cases, the control system precipitated less than 10% of the isotope coprecipitable with the carrier system specific for 'e-globulins. All 'yglobulin values were corrected for radioactivity coprecipitated in the control system. For the determination of sialic acids, the relevant precipitates were washed twice with 2.0 ml of 0.15 NaCl-0.01 Ml phosphate, ph 7.0, and once with 3.0 ml of 5% trichloroacetic acid at 4. The precipitate was resuspended in 3.0 ml of 5% trichloroacetic acid and incubated for 15 min at 800. This procedure quantitatively liberated the sialic acid present in 'y-globulin. The sample was cooled to 40, centrifuged at 9000 X g for 10 min, and the resultant supernatant fluid was set aside. The precipitate was washed once with 546

2 VOL. 59, 1968 BIOCHEMISTRY: SWENSON AND KERN ml of cold 5% trichloroacetic acid, centrifuged, and the supernatant fluid was combined with the above. The combined supernatant fluids were extracted with ether to remove trichloroacetic acid and dried under reduced pressure over KOH. The sample was dissolved in H20, applied to DEAE-paper, and eluted with H20 to remove neutral sugars and hexosamines. Sialic acids were then eluted with 1.0 N HCOOH, dried in a rotary evaporator at 370, dissolved in 1.0 ml Hyamine lox, and counted. The recovery of sialic acid was 80%. To verify the specificity of the assay, formic acid elution was carried out at 40 to minimize deterioration of sialic acid. The eluate was extracted with ether, taken to dryness under reduced pressure over KOH, and chromatographed using the procedure of Svennerholm.7 It was observed that 92% of the label was coincident with N-glycolyl sialic acid and 8% with N-acetyl-neuraminic acid. The evidence that H3-glucosamine and HI-galactose are incorporated solely into the carbohydrate moiety of 7-globulin has been presented previously.2 4,5-H'-L-leucine, and CL-amino acid mixture (uniformly labeled) were purchased from New England Nuclear Corp., and had specific activities of 5 me per Mmole and 1.0 mc per mg, respectively. H'-1-galactose, 1.8 me per jsmole, and H'-6-glucose, 5.0 me per inmole were obtained from Nuclear-Chicago Corp. Generally labeled H'-glucosamine, 3.7 me per smole, was purchased from International Chemical and Nuclear Corp., City of Industry, Calif. HI and C14 were counted with an efficiency of 15% and 70%, respectively. Results.-The time course of appearance of specifically coprecipitated intracellular and extracellular a-globulin derived from cells labeled in vitro with isotopic amino acids or monosaccharides is presented in Figure 1. The results obtained with H3-leucine and a C'4-amino acid mixture confirm previous observations made with antidinitrophenyl antibody4 in showing that intracellular -y-globulin was detectable without delay, while some 20 minutes were required before the extracellular appearance of such label. The differences in the gross shape of the curves, shown for H3-leucine and C'4-amino acids, were expected in view of the incubation conditions that were required in order to make this comparison. In the case of the C'4-amino acids, both glucose and unlabeled amino acids were omitted from the incubation medium. Thus the decline of the curve for intracellular y-globulin at 60 minutes (C 14-amino acids) was probably due to depletion of energy and carbon sources. Under the conditions used for these experiments, the labeled monosaccharides were incorl)orated solely into the carbohydrate moiety of y-globulin.2 It is clear thatill regard to the secretory laghl-galactose incorporation into 7- globulin showed the same pattern of behavior as observed with amino acids. The data for H3-glucosamine incorporation was complicated by the fact that there was a delay in the intracellular appearance of labeled 7-globulin. This delay is probably due to the relatively slow rate of entry of glucosamine into cells as well as the time necessary for equilibration of the isotope with the various pools of precursors, including the uridine diphospho-n-acetyl glucosamine required for glycoprotein synthesis. However, it is clear that the linear portion of the curves for intracellular and extracellular 7-globulin labeled with H'-gluosamine are essentially parallel and are separated by an interval of approximately 20 minutes. Since there are 3 moles of galactose and 8 moles of glucosamine per 9 mole ofy-globulin,8' it was not possible from this data to distinguish between an early rapid synthesis or a slow acquisition of the total carbohydrate during the period of the secretory lag.

3 548 BIOCHEMISTRY: SWENSON AND KERN PRoc. N. A. S. C.P. M. 16,000-12,000 H3_ Leucine i}c4-amino Acids 8,000 a 0 4, H-3-Glucosajmine H3-GlaIctose a 0 _j~~ MINUTES FIG. 1.-Time course of appearance of intracellular and extracellular e-globulin derived from cells incubated with radioactive amino acids or monosaccharides. Radioactivity in intracellular 7y-globulin (@-_); radioactivity in extracellular y-globulin (OO). Lymph node cells were incubated in 30-ml beakers containing 3.0 ml of medium, 5 X 108 cells plus the appropriate isotope. For each time point the contents of a single beaker was used. The amount of isotope used, per 3.0 ml of media, was: for leucine incorporation, 30.uc H3-leucine; for amino acid incorporation, 30 pc C04-amino acid mixture; for glucosamine incorporation, 100,uc HI-glucosamine; and for galactose incorporation, 50,uc H3-galactose. After incubation for the appropriate time the cells were harvested by centrifugation at 1350 X g for 5 min. The extracellular medium was transferred to a tube containing 100 Mmoles of the C12 or H2 form of the compound used for incorporation in 2 ml 0.1 M Tris-HCl, ph 7.4, and centrifuged at 9000 X g for 5 min to remove any particulate matter. The cell pellet was resuspended in 1.0 ml of harvest media containing sodium deoxycholate at a concentration of 0.5% and incubated for 10 min at 3. The volume was then brought to 12.5 ml with 0.15 NaCl-0.01 M phosphate, ph 7.0, containing 100 pmoles of the Ci2 or H2 form of the compound used for incorporation and the tubes centrifuged at 100,000 X g for 60 min to remove insoluble materials. The extracellular and intracellular fractions were then analyzed by the coprecipitation technique as described in Materials and Metds. For several reasons the sialic acid which is known to be in y-globulin affords distinct advantages in determining whether the acquisition of the carbohydrate is rapid or slow: (a) there is 1 mole of sialic acid per mole of y-globulin;8 (b) sialic acid, when present, is a terminal residue of the carbohydrate chain of almost all known glycoproteins; and (c) the relatively gentle acid hydrolysis procedure required for the liberation of sialic acid from y-globulin is identical to that necessary for other sialic acid-containing glycoproteins. If the acquisition of the single residue of sialic acid occurred during the latter part of the secretory lag, it would be expected that the curves for the initial appearance of intracellular and extracellular sialic acid would be close together in time; i.e., markedly less than the 20-minute interval observed with glucosamine and galactose. As demonstrated in Figure 2, the curves for the initial appearance of the intracellular and extracellular sialic acid of 7-globulin are superinmposable.

4 VOL. 59, 1968 BIOCHEMISTRY: SWENSON AND KERN o MINUTES FIG. 2.-Time course of appearance of H3-sialic acid in intracellular and extracellular y-globulin. Radioactivity in intracellular -y-globulin (@-O); radioactivity in extracellular y-globulin (0-C). The procedures used were as described in Fig. 1 except for the following modifications: (1) the contents of two 30-ml beakers were used to determine each time point; (2) 500 yc H3- glucose was added to each flask; (3) the soluble intracellular and extracellular fractions were dialyzed 18 hr vs NaCl M phosphate, ph 7.0, prior to coprecipitation; and (4) Hs-sialic acid was identified and analyzed for as described in Materials and Methods. Evidence that some of the carbohydrate other than sialic acid is also acquired relatively close to the moment of secretion of 7-globulin is presented in Figure 3. In this experiment, H3-glucose was used to label the over-all carbohydrate component, and the sialic acid was then removed from the specifically coprecipitable 7y-globulin prior to isotopic analysis. It can be seen that the labeled carbohydrate in extracellular y-globulin appeared very early in time, since the line drawn to fit these data extrapolates to the origin.'0 Discussion.-The similarity of the kinetics of appearance of intracellular and extracellular 7-globulin, regardless of whether it is labeled in the protein moiety with amino acids or in the carbohydrate moiety with glucosamine or galactose, demonstrates that at least part of the carbohydrate is acquired close to the time of synthesis of the polypeptide chain. It has been suggested that some liver glycoproteins acquire a portion of their glucosamine before the nascent polypeptide chains are completed."i A similar suggestion has been made for the synthesis of immunoglobulins by mouse myeloma cells, purely on the basis of the presence of labeled glucosamine in nascent polypeptide chains derived from polyribosomes.'2 It should be stressed that the immunoglobulins are most probably only one class of many cellular glycoproteins which contain glucosamine and that, in the latter report, the product was not identified as an immunoglobulin. Indeed, of the deoxycholate-soluble materials extracted from rabbit lymph

5 5505BIOCHEMISTRY: SWENSON AND KERN PRoc. N. A. S. ay MINUTES FIG. 3.-The time course of appearance of -y-globulin in the intracellular and extracellular fractions of lymph node cells incubated with H3-glucose. Radioactivity in intracellular y-globulin (@-4); radioactivity in extracellular -y-globulin (0-O). The procedures used were as described in Fig. 1 with 250 juc HI-glucose added per 3.0 ml of incubation media. It should be noted that in the processing of these precipitates their incubation in 5% trichloroacetic acid at 950 for 10 min removes the sialic acid (see Materials and Methods). node cells incubated with H3-glucosamine, only 3 per cent of the trichloroacetic acid-precipitable isotope was detected as y-globulin. Our data appear to be more compatible with the view that the carbohydrate moiety is acquired subsequent to the synthesis of the polypeptide chains. Although the bulk of the 7-globulin labeled in its protein moiety is found in the microsomes, less than 5 per cent of the y-globulin labeled with glucosamine is present in this fraction.2 Since there is evidence for two carbohydrate chains linked directly to y-globulin via glucosamine; , 14 it would be expected that a greater percentage of the carbohydrate label would be detected in the microsome fraction if glucosamine were acquired by the nascent polypeptide chain. Thus, rather than 5 per cent being observed in the microsome fraction, per cent would be found if 2 of the 8 or 9 residues of glucosamine per mole of y-globulin were added before the polypeptide chains were completed. Not only is the microsome fraction relatively free of carbohydrate, but it appears to be relatively free of 7-globulin subunits or their nascent polypeptides, since the specificially coprecipitable isotopic material was essentially equally coprecipitable with anti-7-globulin, antilight chain and antiheavy chain sera. Finally, while we are unable to measure the concentration of the nascent polypeptides, it would be expected that they exist in catalytic quantities relative to the amount of 7- globulin and its subunits. For these reasons we suggest that the carbohydrate is acquired following the completion of synthesis of the polypeptide chains.

6 VOL..59, 19fi8 BIOCHEMISTRY: SWENSON AND KERN551 The observation that H3-sialic acid appeared simultaneously ill both the intracellular and extracellular y-globulin derived from cells incubated in the presence of H3-glucose indicates that this terminal residue of the carbohydrate chain is added immediately prior to secretion. When cells were incubated with H'- glucose and the sialic acid removed from y-globulin prior to isotopic analysis, the label appeared extracellularly without delay, indicating that other carbohydrate residues are added shortly before secretion. Thus, this study of the temporal relationships for the acquisition of the carbohydrate moiety of -y-globulin demonstrates that the complete carbohydrate chain is slowly synthesized over the 15- to 20-minute period which comprises the secretory lag. 5 The data presented herein and in our previous reports'-3 provide a basis for constructing an over-all mechanism for the secretion of -y-globulin. We visualize that newly synthesized heavy and light subunits of 'y-globulin, either separately or in associated form, rapidly enter the cisternae of the endoplasmic reticulum where the bulk of the y-globulin is confined."'6 A priori, the compartmentalization of y-globulin molecules in the cisternal space requires that the exit of molecules from within this structure be restricted. Since almost all of the carbohydrate labeled y-globulin is always found in the particle-free cell sap2 under a variety of conditions used for homogenization and cell lysis, we tentatively suggest that the y-globulin, by an undefined specific process, leaves the cisternal space of the endoplasmic reticulum and enters the cytoplasm which surrounds this structure. Here the carbohydrate chain is acquired slowly over a 15- to 20-minute period and upon its completion the a-globulin is secreted into the extracellular medium. The intracellular site at which the carbohydrate residues are added is at present unclear. The presence of a small amount of carbohydrate-labeled y-globulin in the microsome fraction could be due either to an artifact of the homogenization procedure or to a specific association with this fraction. In the latter case, because of the known heterogeneity of the microsome fraction, one can envision more than one possible site of association. For example, the carbohydratelabeled y-globulin could be present within the same compartment as that known for the bulk of the amino acid-labeled y-globulin.2 If this is so, it is intriguing to consider that the addition of some carbohydrate provides the y-globulin with the specificity required for rapid exit from this compartment. Alternatively, since at least some of the enzymes involved in the transfer of sugars to y-globulin are known to be associated with the microsome fraction,'7 the label detected in this fraction could represent enzyme-,y-globulin complexes existing at the moment of homogenization. It is necessary that any mechanism suggested for the secretion of y-globulin account for the observations of Helmreich, Kern, and Eisen4 who demonstrated that the secretory lag was due to an "ordered" secretion in which older molecules leave the cell before those synthesized more recently. Thus, the absence of apparent mixing of intracellular y-globulins inherent in "ordered" secretion can be accounted for by the slow acquisition of the carbohydrate moiety over the time period of the secretory lag. The presence of y-globulins in various stages of completion would provide for the absence of mixing even though these struc-

7 552 BIOCHEMISTRY: SWENSON AND KERN PROC. N. A. S. turally dissimilar molecules were present in a common pool, i.e., in the cytoplasm surrounding the endoplasmic reticulum. Except for the initial steps concerning the entry of secretory protein into the cisternal space of the endoplasmic reticulum, the mechanism suggested for 'y-globulin secretion by lymph node cells differs markedly from that suggested for the secretory process of pancreatic exocrine cells.18' 19 It is by no means unexpected that the secretory mechanism for these two tissues would be quite distinctive since the cells involved differ in cell architecture and in the nature of the secretory products. In exocrine cells the endoplasmic reticulum is confined to the basal half of the cell adjacent to the basement membrane with zymogen granules clearly defined in the apical portion. Normal rabbit plasma cells have a diffuse endoplasmic reticulum and are devoid of any apparent secretory granules. In addition, the products secreted by exocrine cells:20 e.g., RNase, amylase, because of their degradative enzymatic activity, could pose serious problems to cellular integrity if they freely mixed with the nonsecreted cellular constituents. On the other hand, y-globulin, the sole secretory product of plasma cells,6 is enzymatically inert. We have suggested that the bulk of the carbohydrate is acquired during the transit of y-globulin through the cytoplasmic matrix because it is the simplest mechanism permissible from our data and from available electron microscopic observations. Although the suggested mechanism implies that the acquisition of the carbohydrate is required for secretion, attempts to dissociate the synthesis of protein from the acquisition of the carbohydrate, in order to definitively demonstrate such a role, have been thus far unsuccessful. Summary.-The temporal relationship between the acquisition of the carbohydrate component of 7-globulin and the minutes required for the intracellular transit of this protein has been examined. It was demonstrated that the first carbohydrate residues are added close to the time of synthesis of the polypeptide chains, while the last residues are added immediately prior to secretion of the 7y-globulin into the extracellular medium. A mechanism of secretion is tentatively suggested on the basis of these findings. * Supported by an Advanced Research Fellowship from the American Heart Association. 1Swenson, R. M., and M. Kern, these PROCEEDINGS, 57, 417 (1967). 2 Swenson, R. M., and M. Kern, J. Biol. Chem., 242, 3242 (1967). 3 Kern, M., and R. M. Swenson, in Cold Spring Harbor Symposia on Quantitative Biology, vol. 32, in press. 4 Helmreich, E., M. Kern, and H. N. Eisen, J. Biol. Chem., 236, 464 (1961). 6 Kern, M., E. Helmreich, and H. N. Eisen, these PROCEEDINGS, 45, 862 (1959). 6 Helmreich, E., M. Kern, and H. N. Eisen, J. Biol. Chem., 237, 1925 (1962). 7 Svennerholm, E., and L. Svennerholm, Nature, 181, 1154 (1958). 8 Fleischman, J. B., R. R. Porter, and E. M. Press, Biochem. J., 88, 220 (1963). 9 Nolan, C., and E. L. Smith, J. Biol. Chem., 237, 446 (1962). 10 The precise reason for the constancy of the slope of this extracellular curve in comparison to the changing slopes that are observed using Hs-glucosamine or H'-galactose as labeled indicator is, at present, unknown. Without a clear knowledge of the differences in the rate of equilibration of H-glucose with the pools of the several precursors required for the synthesis of the carbohydrate component of -y-globulin, it is not possible to predict whether one or many slopes should be expected. 11 Lawford, G. R., and H. Schacter, J. Biol. Chem., 241, 5408 (1966). 12 Melchers, F., and P. Knopf, in Cold Spring Harbor Symposia on Quantitative Biology, vol. 32, in press.

8 VOL. 59, 1968 BIOCHEMISTRY: SWENSON AND KERN UtSumi, S., and F. Karush, Biochemistry, 4, 1766 (1965). 14 Smyth, D. S., and S. Utsumi, Nature, 216, 332 (1967). 15 Melchers and Knopf (see ref. 12) have suggested that the acquisition of the total carbohydrate moiety of the immunoglobulin secreted by mouse myeloma cells requires at least 8 min. This suggestion is based on peptide mapping of tryptic digests of a mixture of extracellular and intracellular light chain, labeled with indicator H'-leucine or C14-leucine, respectively. Using digests of intracellular light chains isolated 2, 5, and 8 min after incubation with H3-leucine, they were unable to find H3-label in those map positions known for carbohydratecontaining peptides. Therefore, they concluded that the carbohydrate chain synthesis requires more than 8 min. Since they did not present any evidence showing that such carbohydratecontaining peptides were ever found, we find their conclusion unwarranted. For example, if they were unable to detect such peptides from digests of intracellular light chains after 80 or 800 min of incubation with H3-leucine, they would be compelled logically to conclude that the acquisition of the carbohydrate chain required an unreasonably lengthy period of time. Our experience with rabbit lymph node cells would indicate that only a small percentage of the -yglobulin with completed carbohydrate chains exist intracellularly. Therefore, assuming a similarity between the behavior of rabbit lymph node ahd mouse myeloma cells, the percentage of light chains containing complete carbohydrate chains could be below the level of detectability by their procedure. 16 de Petris, S., G. Karlsbad, and B. Pernis, J. Exptl. Med., 117, 849 (1963). 17 D'Amico, R. P., and M. Kern, unpublished observations. 18 Jamieson, J. D., and G. E. Palade, J. Cell Biol., 34, 577 (1967). 19Jamieson, J. D., and G. E. Palade, J. Cell Biol., 34, 597 (1967). 20 Siekevitz, P., and G. E. Palade, J. Biophys. Biochem. Cytol., 4, 203 (1958).