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Retrograde axonal transport of asialoglycoproteins in mouse trigeminal neurons in vivo and in rat dorsal root ganglia neurons in vitro
14
f¢ram R~'sear{'h,3~7 t t ~8~i} 14 - i~ [~!scvic~
BRE 11483
Retrograde Axonal Transport of Asialoglycoproteins in Mouse Trigeminal Neurons In Vivo and in Rat Dorsal Root Ganglia Neurons In Vitro MARIE GUSTAFSSON, GC)RAN ANDERSSON, ANDERS HJERPE and KRISTER KRISTENSSON
Department of Pathology, Karolinska lnstitutet, Huddinge University Hospital, S-141 86 Huddinge (Sweden) (Accepted July 3rd, 1985)
Key words: asialoglycoprotein- - retrograde axonal transport - - endocytosis - - receptor - - endogenous lectin - trigeminal neuron - - dorsal root ganglia neuron - - mouse
Different terminal sugars of the glycoprotein orosomucoid were exposed by sequential glycosidase digestions. The orosomucoid and its different derivatives were conjugated to horseradish peroxidase by a two-step glutaraldehyde coupling procedure, injected into the snout of 12-day-old mice or exposed to dorsal root ganglia neurons from embryonic rats, cultivated in a two chamber system. A marked increase in transport of the conjugates in the trigeminal and dorsal root ganglia neurons was observed histochemically after removal of sialic acid, exposing galactose as the terminal sugar. Quantitative hydrolysis of galactose residues resulted in reduced uptake, The data suggest the presence of a galactose-recognition molecule in the axon-terminal membrane, involved in retrograde axonal transport.
INTRODUCTION R e t r o g r a d e axonal transport of macromolecules provides a mechanism by which biologically active substances can be translocated to nerve cell bodies subsequent to their adsorption or binding to axon terminals 16. E x a m p l e s of substances taken up by this fashion are nerve growth factor 6, tetanus toxin 27 and certain neurotropic viruses 17. Certain plant lectins specifically recognize c a r b o h y d r a t e units of glycoconjugates at the axon terminal m e m b r a n e , after which a retrograde transport may ensue tl,2v, but, conversely, it is not known if there exist molecules in the axonal m e m b r a n e that can specifically recognize sugar terminals in glycoconjugates present in the environment of the nerve endings. The present study concerns the definition of mechanisms involved in the u p t a k e and r e t r o g r a d e axonal transport of glycoproteins, particularly regarding the existence of so-called e n d o g e n o u s lectins in the neuronal m e m b r a n e . F o r this purpose, the glycoprotein orosomucoid (at-acid glycoprotein) was used as a model glycoprotein since its c a r b o h y d r a t e structure
is well
characterized,
containing
5 asparagine-
linked complex-type bi-, tri- and t e t r a a n t e n n a r y Nglycans per molecule 9.31. Different terminal sugars were exposed by chemical hydrolysis and glycosidase digestions. A m a r k e d preference for retrograde axonal transport was found for the asialoorosomucoid derivative, both in trigeminal neurons in vivo and in in vitro cultured dorsal root ganglia neurons, suggesting the presence of e n d o g e n o u s galactose-specific lectins in the neuronal m e m b r a n e . MATERIALS AND METHODS Orosomucoid (cq-acid glycoprotein) isolated from human plasma was purchased from CalbiochemBehr, C A . The glycosidases were o b t a i n e d from Seikagaku Kogyo, Japan.
Chemical and enzymatic hydrolysis of carbohydrate residues Sialic acid was r e m o v e d from native orosomucoid by hydrolysis in 1 ml. 0.05 M sulfuric acid (1 mg of protein/ml) for 1 h at 80 °C, after which the incubate
Correspondence: G. Andersson, Department of Pathology F42, Huddinge University Hospital, S-141 86 Huddinge, Sweden. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
15 was immediately neutralized with 1 M NaOH and di-
sucrose in the same buffer. The left trigeminal gan-
alyzed against distilled water overnight. The sialic acid content was determined using the resorcinol method of Svennerholm 29. The glycosidase digestions of asialoorosomucoid (1 mg) were done in the presence of 10 nM leupeptin (Sigma, MO), 0.2 mg phenylmethylsulfonyl fluoride (Sigma, MO) in 10 mM phosphate buffer, pH 6.0. Derivative 3 (Fig. 2a, b) was produced by incubation with fl-galactosidase alone; derivative 4 by simultaneous incubation with fl-galactosidase and fl-NAc-hexosaminidase; and finally deritative 5 by incubation with fl-galactosidase, fl-NAc-hexosaminidase and endoglycosidase D; 200 mU each of the glycosidases were used and the incubation time was 24 h at 37 °C. After digestions, the fractions were dialyzed overnight against 0.15 M NaC1 and concentrated using a Diaflo ultrafiitration cell with a PM-10 filter (Amicon, MA). The galactose content of the different derivatives was determined by HPLC after methanolysis according to Hjerpe et al. 13.
glia and sections from the snout were dissected and stored overnight in the sucrose buffer at 4 °C. Frozen sections, 40/~m thick, were cut, rinsed overnight in phosphate buffer and then incubated with sodium nitroferricyanide and tetramethyl benzidine (Sigma, MO), according to the protocol of Mesulam 20. The sections were mounted in glycerin: water 9:1. Six to 7 longitudinal sections through the whole trigeminal ganglion were evaluated. All injections were made under coded numbers and the sections were read blindly. The data are expressed as the mean + S.E.M. of a total of 20 animals analyzed per derivative. The experiment was repeated on 4 occasions.
Preparation of HRP-conjugates Orosomucoid and the 4 different derivatives thus obtained were then conjugated to horseradish peroxidase (HRP) (Sigma, MO) by a two-step-glutaraidehyde coupling procedure, involving the condensation of free aminogroups, e.g. lysine residues in the glycoprotein and in peroxidase with glutaraldehyde 3. The separation of the HRP-conjugate from free HRP was accomplished by filtration on a Sephacryl S-200 column (100 x 1.6 cm) (Pharmacia Fine Chem., Sweden), and monitored by measuring the absorbance at 280 nm and 410 nm in each fraction. Fractions containing the conjugates were concentrated to equal volumes (1 ml) by ultrafiltration, after which the conjugates exhibited equal peroxidase activity as determined spectrophotometrically (data not shown).
In vivo experiment Each of the 5 different conjugates was injected into the left snout of 12-day-old Swiss albino mice (NMRI, ALAB, Sweden) in a volume of approximately 50/~1, representing approximately 50/ag of protein. Twenty-four hours later the mice were perfused through the heart with saline, followed by 1.25% glutaraldehyde and 1% paraformaldehyde in 0.1 M S6rensen's phosphate buffer and by 10% cold
In vitro experiment Dorsal root ganglia (DRG) neurons were obtained from 17-day-old rat embryos (Sprague-Dawley) and cultivated in a two-chamber system 6,32in Petri dishes (Falcon) coated with calfskin collagen (Sigma). The DRGs were dissociated in 5 ml 0.25% Trypsin (Sigma), washed with Eagles minimal essential medium (MEM) (Gibco) and seeded inside the cloning cylinder. The cells were cultivated in Eagles MEM with fetal calf serum (Gibco) (10%) and heat-inactivated horse serum (Gibco) (10%) for two days, and then treated with Eagles MEM containing 10% horse serum and 10 mM cytosine arabinoside (Sigma) for two days. The cells were then refed 3 times/week with Eagles MEM and horse serum. Cells were cultivated at 37 °C in the presence of 5% CO2. Cultures were used for binding and uptake studies 7-10 days after explantation. The different derivatives were diluted with MEM to a final concentration of 3/~M and added to the neurites in the outer chamber. Binding was permitted for 4 h at 4 °C. After extensive washings with MEM, cultures were incubated for 18 h at 37 °C. Following fixation in 1.25% glutaraldehyde + 1% paraformaldehyde in 0.1 M S6rensen's phosphate buffer, pH 7.4, at 4 °C for 2 h; presence of the peroxidase-conjugated glycoproteins in the nerve cell bodies was visualized by the histochemical method using tetramethylbenzidine (TMB) (Sigma) as described by Mesulam20. The ability of neurites to bind and internalize glycoproteins exposing different terminal sugars was assessed by determining the number of peroxidasepositive cell bodies inside the cloning cylinder. The
experiment was performed on a total of 3 cultures per derivative and the experiment was repeated on two occasions. 350
RESULTS
10()
[ i
80 ~
280,
The glycoprotein orosomucoid (cq-acid glycopro-
2101
tein) was used as a model glycoprotein ligand since its
~] i
carbohydrate structure is well characterized, containing 5 asparagine-linked complex-type bi-, tri- and tetraantennary N-glycans per molecule 9,3[. To ex-
604
! ,'I
40 ~
]/
;1 if: , i
,o~ :
pose different terminal sugars, we used mild sulfuric acid hydrolysis, which removes sialic acid residues
1
:
-r
i
l 2
3
4
5
1
2
3
4
5
thereby exposing galactose; the exoglycosidases: fl-galactosidase (C.
Lampas)
which
exposes
N-
acetylglucosamine and fl-NAc-hexosaminidase (C. Lampas) which exposes mannose; and the endoglycosidase D (D. p n e u m o n i a e ) which hydrolyze the remaining N-glycan except for the N-acetyl-glucosamine linked to the asparagine 2,15.
1
2
3
4
5
Fig. 1. Sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern of orosomucoid derivatives treated with mild acid hydrolysis and exo- and endoglycosidases. Lane 1, native orosomucoid; Lane 2, desialylated orosomucoid by treatment of orosomucoid with 0.05 M sulfuric acid at 80 °C for 1 h. Lane 3, orosomucoid was treated as in 2, but in addition incubated with 50 mU fl-galactosidase (C. Lampas) together with 10 nmol leupeptin, 0.2 mg phenylmethylsulfonyl fluoride (PMSF) and 10 mM phosphate buffer, pH 6.0, at 37 °C for 24 h. Lane 4, orosomucoid was treated as in 3, but also with 50 mU flN-acetyl-hexosaminidase (C. Lampas). Lane 5, orosomucoid was treated as in 4, plus with 50 mU endoglycosidase D (D. Pneumoniae). Aliquots corresponding to 10/~g of protein were subjected to electrophoresis on 12.5% polyacrylamide gels containing SDS.
Fig. 2. Endocytosis into trigeminal neurons in vivo (a), and into dorsal root ganglia neurons in culture (b) by derivatives of orosomucoid, treated with mild acid hydrolysis and exo- and endoglycosidases as described in Materials and Methods and Fig. 1. After the treatments, derivatives 1-5 were conjugated to horseradish peroxidase (HRP) by a two-step glutaraldehyde coupling procedure. HRP-glycoprotein conjugates were separated from unreacted HRP by gelfiltration on Sephacryl S-200. In the in vivo experiment (a), around 50/zg of each derivative was injected into the left snout of mice. After 24 h, the animals were perfusion-fixed and the left trigeminal ganglia dissected. Visualization of the peroxidase activity in tissue sections was performed using tetramethylbenzidine (TMB) as described in Materials and Methods. The data are expressed as the number of TMB-positive cells/6-7 sections per ganglion and represent the mean + S.E.M. of 20 ganglia per derivative. In the in vitro experiment (b), around 125/~g of each derivative was added to cultures of dorsal root ganglia neurons prepared and maintained as described in Materials and Methods. The derivatives were added to the outer chamber of dishes and binding was allowed at 4 °C for 4 h. After extensive washing with MEM, cultures were incubated at 37 °C for 18 h. Following fixation, the peroxidase activity was visualized using TMB. The data are expressed as the number of TMB-positive cells/culture dish, and represent the mean _+S.E.M. of 3 cultures per derivative.
SDS-polyacrylamide gel electrophoresis according to Laemmli 18 demonstrated that the glycosidase treatments produced derivatives with slightly increased electrophoretic mobilities, suggesting alterations only in the carbohydrate portion (Fig. 1). Carbohydrate analysis of the different derivatives demonstrated a 90% removal of sialic acid by mild acid hydrolysis (from 0.55 to 0.055/~mol/mg) and 90% hydrolysis of galactose residues (0.44 to 0.04/~mol/mg) by subsequent fl-galactosidase treatment. The content of N-acetylglucosamine and m a n n o s e was not analyzed following further enzymatic digestions. Mild acid hydrolysis rather than n e u r o a m i n i d a s e
17
!~ ~
In vivo experiment
,iii!~ i~~iiiii~ !~i~! ~
~J
b
"
i
The number of labelled neurons in the trigeminal ganglia was counted and the results after injections of the conjugates are given in Fig. 2a. Very few cells had incorporated the native orosomucoid-HRP conjugate. After removal of the sialic acids many more neurons, representing 30% of the total number of counted neurons, were selectively labelled, with an approximately 25-fold increase in number compared to intact orosomucoid. The labelled nerve cell bodies showed a granular reaction product in the cytoplasm (Fig. 3b). Fewer cells were labelled after treatment with the exoglycosidases and after incubation with the endoglycosidase D only very few labelled neurons were seen.
In vitro experiment
f-
A marked increase in accumulation was found after mild acid hydrolysis (Fig. 2b). In this case, about 13% of the total number of counted neurons were labelled. A predominant perinuclear localization of label was noted. This increased uptake of the asialoorosomucoid-HRP derivative was diminished to the control level when galactose was enzymatically removed with fl-galactosidase, and further enzymatic cleavage had no additional effect. DISCUSSION
Fig. 3. Morphological demonstration of neurite transport of native orosomucoid- and asialoorosomucoid-HRP conjugate. A,B: neurons cultivated in vitro were exposed to: A, HRP-orosomucoid; B, HRP-asialoorosomucoid, as decribed in the legend to Fig. 2b. Magnification × 200. C: representative section of trigeminal ganglia from animal injected with HRP-asialoorosomucoid, as described in the legend to Fig. 2a. Note prominent perinuclear staining in positive nerve cell bodies. Magnification x 400.
treatment was preferred for removal of sialic acid residues due to a much lower efficiency of hydrolysis by enzymatic treatment (data not shown).
It is apparent that the removal of sialic acid from the orosomucoid caused an increase in the amount of the conjugate transport to the nerve cell bodies. Several factors may be involved in this alteration, such as changes in the net charge of the molecule by removal of the anionic sialic acids. This possibility is examplifled by the finding that cationized ferritin is transported at much higher rates than anionic 24. Electrostatic effects can, however, not explain the whole increase observed in the present study since further cleavages of asialoorosomucoid, all exposing neutral sugar terminals, considerably reduced the amount of conjugate transport. Alternatively, since removal of terminal sialic acid by mild acid hydrolysis exposes galactose as the terminal sugar, our results indicate that axon terminals have a higher affinity for this sugar. The enhanced uptake of the asialoorosomucoid would then reflect the presence of a galactose-binding molecule on the axon terminal surface. However,
18 in the in vivo experiment (Fig. 2a),/4-galactosidase and fl-NAc-hexosaminidase treatment did not completely abolish uptake. Whether this reflects a lower affinity uptake system for N-acetylglucosamine and monnose-terminated glycoproteins by trigeminal neurons, in contrast to dorsal root ganglia neurons (Fig. 2b), remains to be further explored. Presence of peroxidase-conjugated asialoorosomucoid was noted in 30% of the trigeminal neurons and in around 13% of D R G nerve cell bodies. On one hand, there may be trivial explanations such as limited availability of the conjugates to nerve terminals at the injection site or that the concentration of the conjugates was not optimal. Alternatively, the results may be interpreted as a localization of this galactose-binding protein on a subpopulation of neurons. However, to finally answer this, more detailed immunohistochemical analysis will be required. In recent years, the occurrence of carbohydratebinding proteins has been described in several vertebrate cells (vertebrate lectins) 21.23. In the nervous system, endogenous galactose/lactose binding-proteins have previously been found in developing chick optic tectum 12 and in the electric organ of the electric eellg,30. Lectins with similar molecular mass, isoelectric points, agglutination properties and carbohydrate specificity (mainly G a l f l l - 4 G l c N A c / G a l f l l 4GIc) have been characterized in calf heart a and lung~, mouse 3T3 fibroblasts z6 as well as in chicken intestine 5 and muscle 7,22. In addition, unique galacrose-binding proteins have been demonstrated in mouse 3T3 fibroblasts 26 and rat hepatocytes 1,14 and
REFERENCES 1 Ashwell, G. and Harford, J., Carbohydrate-specific receptors of the liver, Ann. Rev. Biochem., 51 (1982) 531-554. 2 Arakawa, T. and Muramatsu, T., Endo-fl-N-acetylglucosaminidases acting on the carbohydrate moieties of glycoproteins, J. Biochem., 76 (1974) 307-317. 3 Avrameas, S., Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration, Immunohistochemistry, 8 (1971) 1175-1179. 4 Barak-Briles, E., Gregory, W., Fletcher, P. and Kornfeld, S., Vertebrate lectins. Comparison of properties of fl-galactoside-binding lectins from tissues of calf and chicken, J. Cell Biol., 81 (1979) 528-537. 5 Beyer, E.C., Zweig, S.E. and Barondes, S.H., Two lactose binding lectins from chicken tissues, J. Biol. Chem., 255 (1980) 4236-4239.
purified by affinity chromatography on asialoglycoprotein-derivatized agarose. Whereas the calf and chicken lectin may be involved in tissue development, the hepatocyte lectin has been implicated in the clearance of desialylated serum glycoproteins by the liver. Following binding of ligand, exposing galactose/N-acetylgalactosamine, to the receptor protein localized in the plasmalemma, the receptor-ligand complex is internalized and delivered to vesiculo-tubular structures in the periphery of the cell prior to lysosomal degradation of the ligand 1,m. The significance of the presently demonstrated retrograde transport of galactose-terminated glycoproteins in neurons may be of importance for uptake of factors bound to plasma proteins in neurons, and/or in the interaction of envelope glycoproteins of certain neurotropic viruses with the nerve terminals. Further, it is interesting to note that myelin-forming cells express galactocerebrosides on their surface membranes zS. The present suggestion of a high-affinity binding of galactose to axons may therefore prove to be of relevance for glia-axon interactions.
ACKNOWLEDGEMENTS This study was supported by grants from the Swedish Medical Research Council (No. 12X-7141 and No. 12X-4480) and the Research Funds of Karolinska Institutet. We thank Mrs. Inga-Lisa Wallgren for efficient secretarial assistance and Mrs. Mervi Nurminen for technical assistance.
6 Campenot, R.B., Local control of neurite development by nerve growth factor, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 4516-4519. 7 Den, H. and Malinzak, D.A., Isolation and properties of fl-D-galactoside-specific lectin from chick embryo thigh muscle, J. Biol. Chem., 252 (1977)5444-5448. 8 De Waard, A., Hiekman, S. and Kornfeld, S., Isolation and properties of fl-galactoside binding lectins of calf heart and lung, J. Biol. Chem., 251 (1976) 7581-7587. 9 Fournet, B., Montreuil, J., Strecker, G., Dorland, L., Haverkamp, J., Vliegenthart, J., Binene, J.P. and Schmid, K., Determination of the primary structures of 16 asialocarbohydrate units derived from human plasma a-l-acid glycoprotein by 360-MHz ~H NMR spectroscopy and permethylation analysis, Biochemistry, 17 (1978) 5206-5214. 10 Geuze, H.J., Slot, J.W., Strous, Ger J.A.M., Lodish, tt.F. and Schwartz, A.L., Intracellular site of asialoglycoprotein
19 receptor-ligand uncoupling: double-label immunoelectron microscopy during receptor-mediated endocytosis, Cell, 32 (1983) 277-287. 11 Gonatas, N.K., Harper, C., Mizutani, T. and Gonatas, J.O., Superior sensitivity of conjugates of horseradish peroxidase with wheat germ agglutinin for studies of retrograde axonal transport, J. Histochem. Cytochem., 27 (1979) 728-734. 12 Gremo, F., Kobiler, D. and Barondes, S.H., Distribution of an endogenous lectin in the developing chick optic tecturn, J. Cell Biol., 79 (1978) 491-499. 13 Hjerpe, A., Engfeldt, B., Tsegenidis, T. and Antonopoulos, C.A., Separation and determination of neutral monosaccharides using methanolysis and high-performance liquid chromatography, J. Chromatogr., 259 (1983) 334-337. 14 Hudgin, R.L., Pricer, W.E. and Ashwell, G., The isolation and properties of a rabbit liver binding protein specific for asialoglycoproteins, J. Biol. Chem., 17 (1974)5536-5543. 15 Koide, N., Nose, M. and Muramatusu, T., Recognition of IgG by Fc receptor and complement: effects of glycosidase digestion, Biochem. Biophys. Res. Commun., 4 (1977) 838-844. 16 Kristensson, K., Retrograde axonal transport of exogenous macromolecules. In D.G. Weiss and A. Gorio (Eds.), Axoplasmic Transport in Physiology and Pathology, Springer Verlag, Berlin-Heidelberg, 1982, pp. 200-205. 17 Kristensson, K., Implications of axoplasmic transport for the spread of virus infections in the nervous system. In D.G. Weiss and A. Gorio (Eds.), Axoplasmic Transport in Physiology and Pathology, Springer Verlag, Berlin-Heidelberg, 1982, pp. 153-158. 18 Laemmli, U.K., Clevage of structural proteins during the assembly of the head of bacteriophage T4, Nature (London), 227 (1970) 680-685. 19 Levi, G. and Teichberg, V.I., Isolation and physicochemical characterization of electrolectin, a fl-D-galactoside binding lectin from the electric organ of Electrophorus electricus, J. Biol. Chem., 256 (1981) 5735-5740. 20 Mesulam, M.-M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 21 Neufeld, E.F. and Ashwell, G., Carbohydrate recognition
systems for receptor-mediated pinocytosis. In W. Lennarz (Ed.), The Biochemistry of Glycoproteins and Proteoglycans, Plenum Press, New York, 1980, pp. 241-266. 22 Nowak, T.P., Kobiler, D., Roel, L.E. and Barondes, S.H., Developmentally regulated lectin from embryonic chick pectoral muscle, J. Biol. Chem., 252 (1977) 6026-6030. 23 Olden, K., Parent, J.B. and White, S.L., Carbohydrate moieties of glycoproteins. A re-evaluation of their function, Biochim. Biophys. Acta, 650 (1982) 209-232. 24 Olsson, T. and Kristensson, K., Neuronal uptake of iron: somatopetal axonal transport and fate of cationized and native ferritin, and iron-dextran after intramuscular injections, Neuropathol. Appl. Neurobiol., 7 (1981) 87-95. 25 Raft, M.C., Mirsky, R., Fields, K.L., Lisak, R.P., Dorman, S.H., Silberberg, D.H., Gregson, N.A., Leibowitz, S. and Kennedy, M.C., Galactocerebroside is a specific cell-surface antigenic marker for oligodendrocytes in culture, Nature (London), 274 (1978) 813-816. 26 Roff, C.F. and Wang, J.L., Endogenous lectins from cultured cells. Isolation and characterization of carbohydratebinding proteins from 3T3 fibroblasts, J. Biol. Chem., 258 (1983) 10657-10663. 27 Schwab, M.E., Suda, K. and Thoenen, H., Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal transport, J. Cell Biol., 82 (1979) 798-810. 28 Stahl, P., Schlesinger, P., Sigardson, E., Rodman, J.S. and Lee, Y.C., Receptor-mediated pinocytosis of mannose glycoconjugates by macrophages: characterization and evidence for receptor recycling, Cell, 19 (1980) 207-215. 29 Svennerholm, L., Quantitative estimation of sialic acids, Acta Chem. Scand., 12 (1958) 547-554. 30 Teichberg, V.I., Silman, I., Beitsch, D.C. and Resheff, G., A fl-D-galactoside binding protein from electric organ tissue of Electrophorus electricus, Proc. Natl. Acad. Sci. U.S.A., 72 (1975) 1383-1387. 31 Yoshima, H., Matsumoto, A., Mizuochi, T., Kawasaki, T. and Kobata, A., Comparative study of the carbohydrate moieties of rat and human plasma- a~-acid glycoproteins, J. Biol. Chem., 256 (1981) 8476-8484. 32 Ziegler, R.J. and Herman, R.E., Peripheral infection in culture of rat sensory neurons by herpes simplex virus, Infect. Immun. 28 (1980) 620-623.
f¢ram R~'sear{'h,3~7 t t ~8~i} 14 - i~ [~!scvic~
BRE 11483
Retrograde Axonal Transport of Asialoglycoproteins in Mouse Trigeminal Neurons In Vivo and in Rat Dorsal Root Ganglia Neurons In Vitro MARIE GUSTAFSSON, GC)RAN ANDERSSON, ANDERS HJERPE and KRISTER KRISTENSSON
Department of Pathology, Karolinska lnstitutet, Huddinge University Hospital, S-141 86 Huddinge (Sweden) (Accepted July 3rd, 1985)
Key words: asialoglycoprotein- - retrograde axonal transport - - endocytosis - - receptor - - endogenous lectin - trigeminal neuron - - dorsal root ganglia neuron - - mouse
Different terminal sugars of the glycoprotein orosomucoid were exposed by sequential glycosidase digestions. The orosomucoid and its different derivatives were conjugated to horseradish peroxidase by a two-step glutaraldehyde coupling procedure, injected into the snout of 12-day-old mice or exposed to dorsal root ganglia neurons from embryonic rats, cultivated in a two chamber system. A marked increase in transport of the conjugates in the trigeminal and dorsal root ganglia neurons was observed histochemically after removal of sialic acid, exposing galactose as the terminal sugar. Quantitative hydrolysis of galactose residues resulted in reduced uptake, The data suggest the presence of a galactose-recognition molecule in the axon-terminal membrane, involved in retrograde axonal transport.
INTRODUCTION R e t r o g r a d e axonal transport of macromolecules provides a mechanism by which biologically active substances can be translocated to nerve cell bodies subsequent to their adsorption or binding to axon terminals 16. E x a m p l e s of substances taken up by this fashion are nerve growth factor 6, tetanus toxin 27 and certain neurotropic viruses 17. Certain plant lectins specifically recognize c a r b o h y d r a t e units of glycoconjugates at the axon terminal m e m b r a n e , after which a retrograde transport may ensue tl,2v, but, conversely, it is not known if there exist molecules in the axonal m e m b r a n e that can specifically recognize sugar terminals in glycoconjugates present in the environment of the nerve endings. The present study concerns the definition of mechanisms involved in the u p t a k e and r e t r o g r a d e axonal transport of glycoproteins, particularly regarding the existence of so-called e n d o g e n o u s lectins in the neuronal m e m b r a n e . F o r this purpose, the glycoprotein orosomucoid (at-acid glycoprotein) was used as a model glycoprotein since its c a r b o h y d r a t e structure
is well
characterized,
containing
5 asparagine-
linked complex-type bi-, tri- and t e t r a a n t e n n a r y Nglycans per molecule 9.31. Different terminal sugars were exposed by chemical hydrolysis and glycosidase digestions. A m a r k e d preference for retrograde axonal transport was found for the asialoorosomucoid derivative, both in trigeminal neurons in vivo and in in vitro cultured dorsal root ganglia neurons, suggesting the presence of e n d o g e n o u s galactose-specific lectins in the neuronal m e m b r a n e . MATERIALS AND METHODS Orosomucoid (cq-acid glycoprotein) isolated from human plasma was purchased from CalbiochemBehr, C A . The glycosidases were o b t a i n e d from Seikagaku Kogyo, Japan.
Chemical and enzymatic hydrolysis of carbohydrate residues Sialic acid was r e m o v e d from native orosomucoid by hydrolysis in 1 ml. 0.05 M sulfuric acid (1 mg of protein/ml) for 1 h at 80 °C, after which the incubate
Correspondence: G. Andersson, Department of Pathology F42, Huddinge University Hospital, S-141 86 Huddinge, Sweden. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
15 was immediately neutralized with 1 M NaOH and di-
sucrose in the same buffer. The left trigeminal gan-
alyzed against distilled water overnight. The sialic acid content was determined using the resorcinol method of Svennerholm 29. The glycosidase digestions of asialoorosomucoid (1 mg) were done in the presence of 10 nM leupeptin (Sigma, MO), 0.2 mg phenylmethylsulfonyl fluoride (Sigma, MO) in 10 mM phosphate buffer, pH 6.0. Derivative 3 (Fig. 2a, b) was produced by incubation with fl-galactosidase alone; derivative 4 by simultaneous incubation with fl-galactosidase and fl-NAc-hexosaminidase; and finally deritative 5 by incubation with fl-galactosidase, fl-NAc-hexosaminidase and endoglycosidase D; 200 mU each of the glycosidases were used and the incubation time was 24 h at 37 °C. After digestions, the fractions were dialyzed overnight against 0.15 M NaC1 and concentrated using a Diaflo ultrafiitration cell with a PM-10 filter (Amicon, MA). The galactose content of the different derivatives was determined by HPLC after methanolysis according to Hjerpe et al. 13.
glia and sections from the snout were dissected and stored overnight in the sucrose buffer at 4 °C. Frozen sections, 40/~m thick, were cut, rinsed overnight in phosphate buffer and then incubated with sodium nitroferricyanide and tetramethyl benzidine (Sigma, MO), according to the protocol of Mesulam 20. The sections were mounted in glycerin: water 9:1. Six to 7 longitudinal sections through the whole trigeminal ganglion were evaluated. All injections were made under coded numbers and the sections were read blindly. The data are expressed as the mean + S.E.M. of a total of 20 animals analyzed per derivative. The experiment was repeated on 4 occasions.
Preparation of HRP-conjugates Orosomucoid and the 4 different derivatives thus obtained were then conjugated to horseradish peroxidase (HRP) (Sigma, MO) by a two-step-glutaraidehyde coupling procedure, involving the condensation of free aminogroups, e.g. lysine residues in the glycoprotein and in peroxidase with glutaraldehyde 3. The separation of the HRP-conjugate from free HRP was accomplished by filtration on a Sephacryl S-200 column (100 x 1.6 cm) (Pharmacia Fine Chem., Sweden), and monitored by measuring the absorbance at 280 nm and 410 nm in each fraction. Fractions containing the conjugates were concentrated to equal volumes (1 ml) by ultrafiltration, after which the conjugates exhibited equal peroxidase activity as determined spectrophotometrically (data not shown).
In vivo experiment Each of the 5 different conjugates was injected into the left snout of 12-day-old Swiss albino mice (NMRI, ALAB, Sweden) in a volume of approximately 50/~1, representing approximately 50/ag of protein. Twenty-four hours later the mice were perfused through the heart with saline, followed by 1.25% glutaraldehyde and 1% paraformaldehyde in 0.1 M S6rensen's phosphate buffer and by 10% cold
In vitro experiment Dorsal root ganglia (DRG) neurons were obtained from 17-day-old rat embryos (Sprague-Dawley) and cultivated in a two-chamber system 6,32in Petri dishes (Falcon) coated with calfskin collagen (Sigma). The DRGs were dissociated in 5 ml 0.25% Trypsin (Sigma), washed with Eagles minimal essential medium (MEM) (Gibco) and seeded inside the cloning cylinder. The cells were cultivated in Eagles MEM with fetal calf serum (Gibco) (10%) and heat-inactivated horse serum (Gibco) (10%) for two days, and then treated with Eagles MEM containing 10% horse serum and 10 mM cytosine arabinoside (Sigma) for two days. The cells were then refed 3 times/week with Eagles MEM and horse serum. Cells were cultivated at 37 °C in the presence of 5% CO2. Cultures were used for binding and uptake studies 7-10 days after explantation. The different derivatives were diluted with MEM to a final concentration of 3/~M and added to the neurites in the outer chamber. Binding was permitted for 4 h at 4 °C. After extensive washings with MEM, cultures were incubated for 18 h at 37 °C. Following fixation in 1.25% glutaraldehyde + 1% paraformaldehyde in 0.1 M S6rensen's phosphate buffer, pH 7.4, at 4 °C for 2 h; presence of the peroxidase-conjugated glycoproteins in the nerve cell bodies was visualized by the histochemical method using tetramethylbenzidine (TMB) (Sigma) as described by Mesulam20. The ability of neurites to bind and internalize glycoproteins exposing different terminal sugars was assessed by determining the number of peroxidasepositive cell bodies inside the cloning cylinder. The
experiment was performed on a total of 3 cultures per derivative and the experiment was repeated on two occasions. 350
RESULTS
10()
[ i
80 ~
280,
The glycoprotein orosomucoid (cq-acid glycopro-
2101
tein) was used as a model glycoprotein ligand since its
~] i
carbohydrate structure is well characterized, containing 5 asparagine-linked complex-type bi-, tri- and tetraantennary N-glycans per molecule 9,3[. To ex-
604
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40 ~
]/
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,o~ :
pose different terminal sugars, we used mild sulfuric acid hydrolysis, which removes sialic acid residues
1
:
-r
i
l 2
3
4
5
1
2
3
4
5
thereby exposing galactose; the exoglycosidases: fl-galactosidase (C.
Lampas)
which
exposes
N-
acetylglucosamine and fl-NAc-hexosaminidase (C. Lampas) which exposes mannose; and the endoglycosidase D (D. p n e u m o n i a e ) which hydrolyze the remaining N-glycan except for the N-acetyl-glucosamine linked to the asparagine 2,15.
1
2
3
4
5
Fig. 1. Sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern of orosomucoid derivatives treated with mild acid hydrolysis and exo- and endoglycosidases. Lane 1, native orosomucoid; Lane 2, desialylated orosomucoid by treatment of orosomucoid with 0.05 M sulfuric acid at 80 °C for 1 h. Lane 3, orosomucoid was treated as in 2, but in addition incubated with 50 mU fl-galactosidase (C. Lampas) together with 10 nmol leupeptin, 0.2 mg phenylmethylsulfonyl fluoride (PMSF) and 10 mM phosphate buffer, pH 6.0, at 37 °C for 24 h. Lane 4, orosomucoid was treated as in 3, but also with 50 mU flN-acetyl-hexosaminidase (C. Lampas). Lane 5, orosomucoid was treated as in 4, plus with 50 mU endoglycosidase D (D. Pneumoniae). Aliquots corresponding to 10/~g of protein were subjected to electrophoresis on 12.5% polyacrylamide gels containing SDS.
Fig. 2. Endocytosis into trigeminal neurons in vivo (a), and into dorsal root ganglia neurons in culture (b) by derivatives of orosomucoid, treated with mild acid hydrolysis and exo- and endoglycosidases as described in Materials and Methods and Fig. 1. After the treatments, derivatives 1-5 were conjugated to horseradish peroxidase (HRP) by a two-step glutaraldehyde coupling procedure. HRP-glycoprotein conjugates were separated from unreacted HRP by gelfiltration on Sephacryl S-200. In the in vivo experiment (a), around 50/zg of each derivative was injected into the left snout of mice. After 24 h, the animals were perfusion-fixed and the left trigeminal ganglia dissected. Visualization of the peroxidase activity in tissue sections was performed using tetramethylbenzidine (TMB) as described in Materials and Methods. The data are expressed as the number of TMB-positive cells/6-7 sections per ganglion and represent the mean + S.E.M. of 20 ganglia per derivative. In the in vitro experiment (b), around 125/~g of each derivative was added to cultures of dorsal root ganglia neurons prepared and maintained as described in Materials and Methods. The derivatives were added to the outer chamber of dishes and binding was allowed at 4 °C for 4 h. After extensive washing with MEM, cultures were incubated at 37 °C for 18 h. Following fixation, the peroxidase activity was visualized using TMB. The data are expressed as the number of TMB-positive cells/culture dish, and represent the mean _+S.E.M. of 3 cultures per derivative.
SDS-polyacrylamide gel electrophoresis according to Laemmli 18 demonstrated that the glycosidase treatments produced derivatives with slightly increased electrophoretic mobilities, suggesting alterations only in the carbohydrate portion (Fig. 1). Carbohydrate analysis of the different derivatives demonstrated a 90% removal of sialic acid by mild acid hydrolysis (from 0.55 to 0.055/~mol/mg) and 90% hydrolysis of galactose residues (0.44 to 0.04/~mol/mg) by subsequent fl-galactosidase treatment. The content of N-acetylglucosamine and m a n n o s e was not analyzed following further enzymatic digestions. Mild acid hydrolysis rather than n e u r o a m i n i d a s e
17
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In vivo experiment
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b
"
i
The number of labelled neurons in the trigeminal ganglia was counted and the results after injections of the conjugates are given in Fig. 2a. Very few cells had incorporated the native orosomucoid-HRP conjugate. After removal of the sialic acids many more neurons, representing 30% of the total number of counted neurons, were selectively labelled, with an approximately 25-fold increase in number compared to intact orosomucoid. The labelled nerve cell bodies showed a granular reaction product in the cytoplasm (Fig. 3b). Fewer cells were labelled after treatment with the exoglycosidases and after incubation with the endoglycosidase D only very few labelled neurons were seen.
In vitro experiment
f-
A marked increase in accumulation was found after mild acid hydrolysis (Fig. 2b). In this case, about 13% of the total number of counted neurons were labelled. A predominant perinuclear localization of label was noted. This increased uptake of the asialoorosomucoid-HRP derivative was diminished to the control level when galactose was enzymatically removed with fl-galactosidase, and further enzymatic cleavage had no additional effect. DISCUSSION
Fig. 3. Morphological demonstration of neurite transport of native orosomucoid- and asialoorosomucoid-HRP conjugate. A,B: neurons cultivated in vitro were exposed to: A, HRP-orosomucoid; B, HRP-asialoorosomucoid, as decribed in the legend to Fig. 2b. Magnification × 200. C: representative section of trigeminal ganglia from animal injected with HRP-asialoorosomucoid, as described in the legend to Fig. 2a. Note prominent perinuclear staining in positive nerve cell bodies. Magnification x 400.
treatment was preferred for removal of sialic acid residues due to a much lower efficiency of hydrolysis by enzymatic treatment (data not shown).
It is apparent that the removal of sialic acid from the orosomucoid caused an increase in the amount of the conjugate transport to the nerve cell bodies. Several factors may be involved in this alteration, such as changes in the net charge of the molecule by removal of the anionic sialic acids. This possibility is examplifled by the finding that cationized ferritin is transported at much higher rates than anionic 24. Electrostatic effects can, however, not explain the whole increase observed in the present study since further cleavages of asialoorosomucoid, all exposing neutral sugar terminals, considerably reduced the amount of conjugate transport. Alternatively, since removal of terminal sialic acid by mild acid hydrolysis exposes galactose as the terminal sugar, our results indicate that axon terminals have a higher affinity for this sugar. The enhanced uptake of the asialoorosomucoid would then reflect the presence of a galactose-binding molecule on the axon terminal surface. However,
18 in the in vivo experiment (Fig. 2a),/4-galactosidase and fl-NAc-hexosaminidase treatment did not completely abolish uptake. Whether this reflects a lower affinity uptake system for N-acetylglucosamine and monnose-terminated glycoproteins by trigeminal neurons, in contrast to dorsal root ganglia neurons (Fig. 2b), remains to be further explored. Presence of peroxidase-conjugated asialoorosomucoid was noted in 30% of the trigeminal neurons and in around 13% of D R G nerve cell bodies. On one hand, there may be trivial explanations such as limited availability of the conjugates to nerve terminals at the injection site or that the concentration of the conjugates was not optimal. Alternatively, the results may be interpreted as a localization of this galactose-binding protein on a subpopulation of neurons. However, to finally answer this, more detailed immunohistochemical analysis will be required. In recent years, the occurrence of carbohydratebinding proteins has been described in several vertebrate cells (vertebrate lectins) 21.23. In the nervous system, endogenous galactose/lactose binding-proteins have previously been found in developing chick optic tectum 12 and in the electric organ of the electric eellg,30. Lectins with similar molecular mass, isoelectric points, agglutination properties and carbohydrate specificity (mainly G a l f l l - 4 G l c N A c / G a l f l l 4GIc) have been characterized in calf heart a and lung~, mouse 3T3 fibroblasts z6 as well as in chicken intestine 5 and muscle 7,22. In addition, unique galacrose-binding proteins have been demonstrated in mouse 3T3 fibroblasts 26 and rat hepatocytes 1,14 and
REFERENCES 1 Ashwell, G. and Harford, J., Carbohydrate-specific receptors of the liver, Ann. Rev. Biochem., 51 (1982) 531-554. 2 Arakawa, T. and Muramatsu, T., Endo-fl-N-acetylglucosaminidases acting on the carbohydrate moieties of glycoproteins, J. Biochem., 76 (1974) 307-317. 3 Avrameas, S., Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration, Immunohistochemistry, 8 (1971) 1175-1179. 4 Barak-Briles, E., Gregory, W., Fletcher, P. and Kornfeld, S., Vertebrate lectins. Comparison of properties of fl-galactoside-binding lectins from tissues of calf and chicken, J. Cell Biol., 81 (1979) 528-537. 5 Beyer, E.C., Zweig, S.E. and Barondes, S.H., Two lactose binding lectins from chicken tissues, J. Biol. Chem., 255 (1980) 4236-4239.
purified by affinity chromatography on asialoglycoprotein-derivatized agarose. Whereas the calf and chicken lectin may be involved in tissue development, the hepatocyte lectin has been implicated in the clearance of desialylated serum glycoproteins by the liver. Following binding of ligand, exposing galactose/N-acetylgalactosamine, to the receptor protein localized in the plasmalemma, the receptor-ligand complex is internalized and delivered to vesiculo-tubular structures in the periphery of the cell prior to lysosomal degradation of the ligand 1,m. The significance of the presently demonstrated retrograde transport of galactose-terminated glycoproteins in neurons may be of importance for uptake of factors bound to plasma proteins in neurons, and/or in the interaction of envelope glycoproteins of certain neurotropic viruses with the nerve terminals. Further, it is interesting to note that myelin-forming cells express galactocerebrosides on their surface membranes zS. The present suggestion of a high-affinity binding of galactose to axons may therefore prove to be of relevance for glia-axon interactions.
ACKNOWLEDGEMENTS This study was supported by grants from the Swedish Medical Research Council (No. 12X-7141 and No. 12X-4480) and the Research Funds of Karolinska Institutet. We thank Mrs. Inga-Lisa Wallgren for efficient secretarial assistance and Mrs. Mervi Nurminen for technical assistance.
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