Sialic Acid Production by Metabolically Engineered Escherichia coli
Removal of Sialic Acids From the Purified Insulin Receptor r
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Transcript of Removal of Sialic Acids From the Purified Insulin Receptor r
Vol. 129, No. 3, 1985 BIOCHEMICALANDBIOPHYSICALRESEARCH COMMUNICATIONS
June28.1985 Pages 739-745
REMOVAL OF SIALIC ACIDS FROM THE PURIFIED INSULIN RECEPTOR RESULTS IN ENHANCED INSULIN-BINDING AND KINASE ACTIVITIES
Yoke Fujita-Yamaguchi, Yuichi Sato, and Satish Kathuria
Department of Molecular Genetics, Beckman Research Institute of the City of Hope, 1450 East Duarte Road
Duarte, California 91010
Received fipril 9, 1985
SUMMARY: Neuraminidase treatment of the purified insulin receptor resulted in an increase in both insulin-bindinq and kinase activities. Neuraminidase- treated 0: and B subunits moved further than native subunits on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The enhancement of insulin-binding and kinase activities and increased mobility of the subunits on SDS-PAGE were not observed when the receptor was treated with neuraminidase in the presence of neuraminidase inhibitor. These results suggest that terminal sialic acid residues have a significant role in insulin-binding and kinase activities. The involvement of sialic acid residues in the activi- ties of the receptor has not been detected by previous studies. QI 1985 Academic Press, Inc.
An intact insulin receptor is composed of a and p subunits with molecular
weight of 125,000 and 90,000, respectively (1). Both subunits are glycoproteins
which appear to have N-asparagine linked oligosaccharide chains (2-4).
Early studies by Cuatrecasas and Illiano suggested that sialic acids at the
cell surface may be involved in the process of signal-transduction from insulin-
binding to glucose transport and lipolysis, but is not involved in the recogni-
tion of insulin by the receptor (5). Since then, the role of terminal
carbohydrate residues in insulin-binding activity has been studied by several
groups (6-8). In general, removal of sialic acids from solubilized or
membrane-bound insulin receptors by neuraminidase has not been shown to
have any effect on insulin-binding activity, while recent studies on the
biosynthesis of the insulin receptor in cultured cells have revealed that
the oligosaccharide chains of the receptor are required for processing of
ABBREVIATIONS SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, P,3-Dehydro NANA: 2,3-Dehydro-neuraminic acid, DTT: Dithiothreitol
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Vol. 129, No. 3, 1985 8lOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the receptor-precursor which leads to the formation of functional insulin
receptors (9,lO).
We have previously purified and characterized the human insulin recep-
tor with high insulin-binding and kinase activities (11-14). Amino acid
analysis of the purified subunits showed significant amounts of N-acetylgluco-
samine, indicating the presence of N-linked carbohydrate chains in both receptor
subunits (14). In the course of our studies on structure-function relationship
of the insulin receptor, we have investigated the role of terminal carbohydrate
residues using our purified receptor.
MATERIALS AND METHODS: Neuraminidase Type X from Clostridium perfringens (150 units/mg protein) and @-galactosidase grade IX from E. coli (630 units/mg protein) were purchased from Sigma. Crystalline porcine insulin was kindly supplied by Eli Lilly. tion site of pp60src
A synthetic peptide resembling the tyrosine phosphoryla- (Arg-Arg-Leu~Ile-G~~~Asp-Ala-Glu-Tyr-Ala-Ala-A~~-Gly) was
purchased from Peninsula Laboratories. I-Labeled insulin and [r- PI-ATP were from New England Nuclear. 2,3-Dehydro-neuraminic acid was generated by saponifying its methyl ester which was generously supplied by Dr. S.W. Tanenbaum (College of Environmental Science and Forestry, State University of New York, Syracuse, NY) (15). Molecular weight markers (myosin, B-galactosidase, phosphorylase b, bovine albumin and ovalbumin) were obtained from Bio-Rad. All other chemicals used were reagent grade.
Insulin receptor was purified 2400-fold with a yield of 409: from human pla- cental membranes by sequential affinity chromatography on wheat germ agglutinin- and insulin-Sepharose columns as described previously (11).
The purified insulin receptor (-0.1 pg) was treated with neuraminidase (0.5 - 10 pg/ml) and/or B-galactosidase (100 and 1000 pg/ml) in 20 ~1 of 50 mM Tris-HCl buffer, pti 7.4, containing 0.1% Triton X-100 at 37°C for 1 h. After the reaction, an aliquot (6 ~1) was assayed for insulin-binding activity as pre- viously described (11). Insulin receptor incubated under the same conditions without the enzyme was also assayed as a control. Specific insulin-binding activity of the enzyme-treated receptor was expressed as percent of that of the control.
In order to examine the effect of neuraminidase treatment on kinase acti- vity in the absence and presence of insulin, an exogeneous substrate was used.
for 8 h f ' The puri ied receptor (-0.1 pg) was treated with neuraminidase (5 pg/ml) at 4" C
and incubated with 1 pM of insulin at 4°C overnight in 20 ~1 of 50 mM Tris-HCl buffer, pH 7.4, containing 0.12 Triton X-100. Added to this solution was 5 ~1 of 50 mM Tris-HCl buffer, pH 7.4, containing 12 mM MnC12, 90 mM MgCl2 la;dazdi:go: ~:e,:r~;o'e~~t~dr_,,,tide. The phosphorylation reaction was initiated
PI-ATP (5-10 &i/nmol) and carried out for 40 min at room temperature. The reaction was terminated by the addition of 50 ,,l of 5x trichloroacetic acid and 20 ~1 of bovine serum albumin (10 mg/ml). After incu- bating this solution at 0°C for 30 min, the proteins were precipitated by centrifugation for 5 min. A 35 ~1 aliquot of the supernatant was spotted on a piece of phosphocellulose paper (Whatman, P81). The paper was extensively
1 We have used two different conditions for neuraminidase treatment in this study, since the kinase activity is found to be very unstable at 37'C. As far as the binding activity is concerned, effects of the enzyme treatments under both conditions (37“C, 1 h, and 4"C, 8-14 h) were the same.
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Vol. 129,No. 3, 1985 BlOCHEMlCALAND8lOPHYSlCALRESEARCHCOMMUNlCATlONS
washed in 75 mM phosphoric acid and placed in a vial containing Aquasol (New England Nuclear). Duplicate of papers were prepared from each reaction mixture and 32P incorporated into the peptide was quantitated in a liquid scintillation counter. The kinase activity of neuraminidase-treated receptor was expressed as percent of that of the receptor without the enzyme-treatment.
RESULTS
The effect of neuraminidase treatment on insulin-binding activity of the
purified receptor was examined. As summarized in Table 1, insulin-binding acti-
vity was significantly enhanced after the receptor was treated with neuramini-
dase (2-10 ug/ml) at 37°C for 1 h. Neither 5-galactosidase treatment of the
intact receptor nor that of neuraminidase treated-receptor affected its insulin-
binding activity (data not shown).
In order to determine the effect of neuraminidase treatment on kinase acti-
vity, the enzyme-treated receptor was assayed by phosphorylation of the
src-related peptide (Table 2). The peptide assay revealed that neuraminidase-
treated receptor exhibits higher kinase activity than the receptor without the
enzyme treatment in the absence and presence of insulin (Table 2). These
results suggest that neuraminidase treatment of the purified insulin receptor
enhances both insulin-binding and kinase activities.
SDS-PAGE analysis of the receptor after neuraminidase treatment revealed
that the mobility of the enzyme-treated a and 5 subunits was increased as pre-
viously observed by others (16-17) (Fig. 1, lane l-4).
TABLE 1 The effect of neuraminidase treatment on insulin-binding activity of the
purified insulin receptor
Neuraminidase
w/ml
Insulin-binding activity(a) %
Average + SD No. of Experiments
0 100
1 126.5 + 20.0 4
2 143.0 2
5 150.8 + 14.7 9
10 168.4 + 18.6 5
(a)Insulin-binding activity was measured at a concentration of 0.8ng of 1251-insulin/ml as previously described (11).
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Vol. 129, No. 3, 1985 8lOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS
TABLE 2
The effect of neuraminidase on kinase activity of the purified insulin receptor in the absence and presence of insulin
Neuraminidase Insulin
a/ml !JM
Kinase activity(a)
Average + SD No. of Experiments
0 0 100
(a) Kinase activity was measured using the synthetic peptide resembling the autophosphoyrlation site of pp60SrC as described in Methods.
(b) Insulin stimulated kinase activity an average 3.1-fold.
To determine whether or not neuraminidase itself is really responsible for
enhancement of the insulin-binding and kinase activities and increased mobility
of the subunits on SDS-PAGE, the neuraminidase inhibitor, 2,3-dehydro-neuraminic
123456789
i (II Mrx 10m3
- 200
- 116
* ',% 3-l i p= ^i I^ - 92
- 66
- 45
Neuraminidase (Pgl ml 1 012512550
23'7e;gy NANA 0 0 0 0 2 2 4 8 8
Fig. 1.. The effects of neuraminidase and neuraminidase plus inhibitor on the mobilltles of insulin receptor subunits on SDS-PAGE. The purified insulin receptor (-4l.5 ug) was incubated at 4°C for 14 hr with no addition (lanes 1,9) 1 ug/ml (lanes 2,5), 2 ug/ml (lanes 3,6) and 5 ug/ml (lanes 4,7,8) of neuramini- dase, in the absence (lanes 1,2,3,4) and presence (lanes 5,6,7,8,9) of 2.3-dehydro NANA (lanes 5,6; &M, lane 7; 4mM, lanes 8,9; 8mM). Each sample was analyzed by SDS-PAGE (18) under reducing conditions, and stained with silver.
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Vol. 129, No. 3, 1985 BIOCHEMICALAND BIOPHYSICALRESEARCH COMMUNICATIONS
TABLE 3
The efflect of neuraminidase inhibitor on insulin binding (a) and kinase activity (b) of the purified insulin receptor during neuraminidase treatment
Neuraminidase (us/ml)
P,3-Dehydro neuraminic acid
(mM) 0 1 5
(a) 0 100 125 147 0.1 --- ;; 149 1.0 --- 125 2.0 95 87 113
(b) 0 100 174 8 103 106
(a)The binding assay was done in duplicate. The mean values are listed. The two individual values did not differ by more than 107 from the mean value.
(b)Kinase activity was measured using the Src-related peptide in the absence of insulin as described in Methods.
acid (2,3-dehydro NANA) was incubated together with the enzyme. The neuramini-
dase inhibitor prevented the effect of neuraminidase treatment on insulin-
binding and kinase activities (Table 3). An increase in the electrophoretic
mobilities of both a and B subunits was also prevented by the presence of
2,3-dehydro NANA (Fig. 1, 5-9). These studies strongly suggested that terminal
sialic acid residues have a significant role in both insulin-binding and kinase
activities of the insulin receptor, and excluded the possibility that proteoly-
tic contaminants from the neuraminidase preparation induced such changes.
DISCUSSION
The role of terminal carbohydrate residues in insulin binding has been
studied by several groups (5-8). In these studies, membrane-bound or
solubilized receptor preparations were treated with glycosidases. In summary,
glycosidase treatment did not affect the binding activity (5-7) or decreased the
activity (8). In contrast to these studies, we have found, using purified insu-
lin receptor, that neuraminidase treatment enhances insulin-binding activity.
Scatchard analyses (19) of the receptor with or without neuraminidase treatment
indicated that the enhanced insulin-binding activity was caused by affinity
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Vol. 129, No. 3, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
changes (data not shown). In some previous studies (5,7,8), since no evidence
was shown to indicate that the glycosidases were active on the receptor, the
lack of an effect of neuraminidase treatment on insulin-binding activity could
be explained by incomplete digestion of the receptor. On the other hand, Kasuga
et al. showed the evidence for removal of sialic acid residues from the insulin
receptor, but they still did not observe any changes in insulin-binding acti-
vity (6). The reason for their results could be explained by a contamination of
the neuraminidase preparation with proteases which could cancel the effect of
neuraminidase treatment. The difference between our results and previous
reports is most likely to be attributed to the purity of the assay system. The
present study using purified receptor should also be distinguished from somewhat
similar studies using intact cells, since protein synthesis and degradation are
not involved in our assay system.
Effects of the sialic acid removal on tyrosine-specific protein kinase
activity have never been reported. Our present study using purified receptor
revealed that sialic acid removal enhances the kinase activity to a similar
extent as is observed with insulin-binding activity. Preliminary Lineweaver-
Burk plot (20) analysis on the kinase activity of the receptor with or without
neuraminidase treatment showed an increase in the Vmax for the peptide after the
treatment.
The degree of enhancement in the receptor activities could be changed if
heterogeneity in terms of sialic acid contents exists in the purified receptor
preparations. In fact, our purified receptor subunits have been shown to have
p1 heterogeneity by O'Farrell's two-dimentional gel analysis (14), which is most
likely caused by heterogeneity in sialic acid contents. Terminal sialic acid
residues could have been removed from a certain population of the receptor mole-
cules during purification or even while the receptors are still in the membra-
nes. Such sialic acid-free receptors would exhibit higher binding and kinase
activities than an intact receptor. Thus, depending on the amount of sialic
acid free-receptors in the preparation, the enhancement of the receptor
activities after neuraminidase treatment is expected to be different. This
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Vol. 129, No. 3, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
would be one of the reasons for rather high errors obtained in the present
experiments. Whether sialic acid residues are directly involved in insulin
binding sites and kinase active sites or in holding a certain conformation of
the receptor molecule is not known. However, it is of particular interest that
sialic acid-free receptor shows an increase in insulin-binding and kinase acti-
vities, which suggests that an intact insulin receptor is not fully active.
ACKNOWLEDGMENT
We thank Dr. S.W. Tanenbaum for a gift of 2,3-dehydro NANA methyl ester, and S. Hartmann for preparing placental membranes. We also thank M. Neermann for typing this manuscript. This work was supported by Grants from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases (AM29770 and AM34427).
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REFERENCES
Czech, M.P., Massague, J., and Pilch, P.F. (1981) Trends Biockem. sci. 5, 222-225. Cuatrecasas, P., and Tell, G.P.E. (1973) Proc. mtZ. ACCZ~. sci. USA, 70, 485-489. Hedo, J.A., Harrison, L.C., and Roth, J. (1981) Biochemistry 20, 3385-3393. Hedo, J.A., Kasuga, M., Van Obberghen E., Roth, J., and Kahn,T.R. (1981) Proc. watt. Acad. SC?:. USA,78, 4791-4795. Cuatrecasas, P., and Illiano, G. (1971) c7. niol. fiem. 246, 4938-4946. Kasuga, M., Ezaki, O., Okanuma, Y., and Kosaka, K. (198rHom. Met&. qes. 12, 494-496. Clark, S., DeLuise, M., Larkins, R.G., Melick, R.A., and Harrison, L.C. (1978) Kockem. ,T. 174, 37-43. Caron, M., Picard, J., and Kern, P. (1978) Riockim. ~iophys. Acta, 512, 29-40. Hedo, J.A., Kahn, C.R., Hayashi, M., Yamada, K.M., and Kasuga, M. (1983) J. ,X02. aem. 258, 10020-10026. Ronnett, G.V., Knutson, V.P. Kohanski, R.A., Simpson, T.L., and Lane, M.D. (1984) .T. R?:OZ mem. 259, 4566-4575. Fujita-Yamaguchi, Y., Choi, S., Sakamoto, Y., and Itakura, K. (1983) ,T. viol. fiem. 258, 5045-5049. Kasuga, M., Fujita-Yamaguchi, Y., Blithe, D.L., and Kahn, C.R. (1983) Proc. VU~Z. Acad. ~ci. USA 80, 2137-2141. Kasuga, M., Fujita-Yamaguchi, Y., Blithe, D.L., White, M.F., and Kahn, C.R. (1983) J. Rio%. &em. 258, 10973-10980. Fujita-Yamaguchi, Y. (1984) 6. niot. &em.=, 1206-1211. Kumar, V., Kessler, J., Scott, M.E., Patwardhan, B.M., Tanenbaum, S.W. and Flashner, M. (1981) Carbohydrates Res. 94, 123-130. Jacobs, S., Hazum, E., and Cuatrecasas, P. (1980) Riockem. ~iophys. Res. corrimm. 94, 1066-1073. Heidenrezh, K.A., Zahniser, N.R., Berhanu, P., Brandenburg, D., and Olefsky, J.M. (1983) ,T. niot. &em. 258, 8527-8530. Laemmli, U.K. (1970) Nature (London) 227, 680-685. Scatchard, G. (1949) AWZ. N.Y. Acad. xi. s, 660-672. Lineweaver, H. and Burk, D. (1934) J. Amer. fiem. SOC. 3: 658-666.
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