Neuroscience Letters, 86 (1988) 213-218 213 Elsevier Scientific Publishers Ireland Ltd.

NSL 05206

Modification of the apparent molecular weight of different benzodiazepine binding proteins from rat

brain membranes by various endoglycosidases

Werner Sieghart and Karoline Fuchs Department of Biochemical Psychiatry, Psychiatrische Universitiitsklinik, Vienna (Austria)

(Received 12 November 1987; Revised version received 14 December 1987; Accepted 14 December 1987)

Key words: Benzodiazepine binding protein; Photoaffinity labeling; Endoglycosidase

Membranes from rat cerebellum or hippocampus were photolabeled by [3H]tlunitrazepam and incu- bated with various endoglycosidases. The largest reduction in apparent molecular weight of photolabeled proteins Psi (mol. wt. 51,000) and P55 (mol. wt. 55,000) was obtained using endoglycosidase F or glycopep- tidase F. However, even after extensive treatment with these enzymes the deglycosylated products of pro- teins Ps~ (apparent mol. wt. 44,000) andP55 (two peptides with apparent mol. wt. 48,000 and 51,000) still differed in their apparent molecular weight. This might indicate that structural features other than N- linked carbohydrates are responsible for the difference in apparent molecular weight of these proteins.

Several lines o f evidence suppor t the existence o f at least two 'central ' benzodiaze- pine receptor subtypes. Thus, it has been demonst ra ted that at least two proteins with

apparent molecular weight 51,000 (Ps0 and 55,000 (P55) are irreversibly labeled by [3H]flunitrazepam when membranes f rom certain brain regions are irradiated with

UV light in the presence o f this radiolabeled ligand [1, 5, 6, 13]. Proteins Psi and P55 seem to be associated with the 'central ' type o f benzodiazepine receptors since irre-

versible labeling o f these proteins was inhibited by 2 /zM diazepam but not by 2/~M Ro 5-4864. The different molecular weight, regional distribution [1, 6, 11, 13], postna- tal development [4, 14] and pharmacological binding properties o f proteins Psi and

P55 [15] support the hypothesis that these proteins are associated with separate and

distinct benzodiazepine receptor subtypes [10]. Other experiments have indicated that benzodiazepine receptors associated with

proteins P51 and P55 are differentially protected against t reatment with trypsin when brain membranes are treated with this protease [3]. One o f the reasons for such a differential protect ion or for a difference in their apparent molecular weight could

Correspondence: W. Sieghart, Department of Biochemical Psychiatry, Psychiatrische Universit/itsklinik, W~hringer Giirtel 18-20, A-1090 Vienna, Austria.

0304-3940/88/$ 03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd.

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be a different carbohydrate content of proteins Psi and P55- In the present study the capacity of several different endoglycosidases to reduce the apparent molecular weight of these proteins was investigated.

Sprague-Dawley rats (200-250 g) were killed by decapitation. Brains were rapidly removed and dissected. Membranes from cerebellum or hippocampus of adult rats were isolated as described previously [3] and were incubated for 90 min at 0°C with 10 nM [3H]flunitrazepam (76.9 Ci/mmol, New England Nuclear) in 2 ml of an incu- bation medium that contained 150 mM NaC1 and 50 mM Tris-citrate buffer, pH 7.1. Samples were then irradiated for 30-60 min with a Camac de Luxe UV lamp (366 nm) at a distance of 10 cm. Membranes were isolated by centrifugation for 10 min at 48,000 g and washed 3 times by resuspension and centrifugation with 5 ml of a 50 mM sodium phosphate buffer, pH 6.0. The membrane pellets were then resus- pended in 150 pl of an incubation buffer containing either 50 mM Tris-citrate, pH 6.1 (for endoglycosidase D), or 50 mM sodium phosphate, pH 6.0 (for endoglycosi- dase F), pH 6.8 (for endoglycosidase H) or pH 8.6 (for glycopeptidase F), in addition to 0.1% sodium dodecylsulfate (SDS), 1% mercaptoethanol, 5 mM EDTA and 0.5 mM phenylmethylsulfonyl-fluoride (PMSF) and were kept in a boiling water bath for 3 min. Two hundred/zl of the same respective incubation buffer containing either no endoglycosidases or 2.5 units endoglycosidase F (EC 3.2.1.96, Boehringer Mann- heim Biochemicals, F.R.G.), 0.02 units endoglycosidase H (EC 3.2.1.96, Genzyme, Boston, MA, or Boehringer, Mannheim), or 0.02 units endoglycosidase D (EC 3.2.1.96, Boehringer Mannheim) were added, and samples were incubated for 17-96 h at 37°C. When glycopeptidase F (N-glycanase, peptide:N-glycosidase F, EC 3.2.2.18; Genzyme, Boston, MA, or Boehringer Mannheim Biochemicals) was used for the removal of carbohydrates, the incubation conditions were exactly as recom- mended in the data sheet for N-glycanase (Genzyme) using 3 units of this enzyme.

In some experiments, fresh endoglycosidase F or H, or glycopeptidase F was added to the respective samples 17 h after the start of incubation, and the samples were incubated for another 17 h with these enzymes. At the end of the incubation, 200 /LI SDS-stop solution (8.33% SDS, 60 mM Tris-Cl, pH 7.4, 3 mM EDTA, 388 mM fl-mercaptoethanol, 75.8 mg/ml saccharose and 0.012 mg/ml Bromophenol blue trac- king dye) was added and after boiling for 2 min, the samples were subjected to SDS- polyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide gels and fluorography [13].

On photolabeling of brain membranes with 10 nM [3H]fiunitrazepam, predomi- nantly one protein (Ps1) was specifically and reproducibly labeled in cerebellum, and at least two proteins (Psi and P55) were labeled in hippocampus (Fig. 1) [1, 5, 6, 13]. Labeling of a protein with apparent molecular weight 44,000 was variable. It was absent in most experiments, but in some experiments this protein was rather strongly labeled. Since labeling of this protein similar to that of proteins Psi and P55 was inhi- bited in the presence of 10 pM diazepam, it is possible that this protein was a degra- dation product of proteins Psi or P55. In contrast, labeling by [3H]flunitrazepam of proteins with apparent molecular weight 33,000 and 26,000 was not inhibited in the presence of 10/tM diazepam, and thus, these proteins seem not to be associated with

Cerebellum I

Additions none F H D

Hippocampus ~ r

none F H D Origin

215

8 0

5 0

3 0

t~ I 0

x

t~ 0 ~

0 @

0

Fig. 1. Fluorography illustrating degradation of different benzodiazepine binding proteins by incubation of photolabeled membranes from cerebellum or hippocampus with various endoglycosidases. Membranes were photolabeled with 10 nM pH]flunitrazepam and were then incubated in the absence or presence of various endoglycosidases as described in the text. Membranes were then subjected to SDS-PAGE and fluorography. F, endoglycosidase F; H, endoglycosidase H; D, endoglycosidase D. The experiment was performed 4 times with essentially similar results.

benzodiazepine receptors (experiments not shown). When photolabeled membranes from cerebellum or hippocampus were incubated

in the presence of endoglycosidase D which so far has received only limited applica- tion due to its high substrate specificity [17], no change in the photolabeling pattern was observed on subsequent SDS-PAGE and fluorography (Fig. 1). However, incu- bation of membranes with endoglycosidase H, which removes N-linked mannose- rich-carbohydrates and certain hybrid oligosaccharides from glycoproteins [19], resulted in a partial disappearance of proteins Pst and P55 and the appearance of a protein with apparent molecular weight 48,000 in both brain regions. Incubation of membranes with endoglycosidase F, which cleaves most high mannose, and some hybrid and complex glycanes from glycoproteins [18] again resulted in a partial dis- appearance of proteins P51 and P55 but the appearance of two photolabeled proteins with apparent molecular weights 44,000 and 48,000. Labeling of the proteins with apparent molecular weight 48,000 (and 51,000) was reproducibly stronger in hippo- campus than in cerebellum. Exactly the same pattern of radiolabeled peptides was obtained when membranes from cerebellum or hippocampus were treated with glyco- peptidase F (experiments not shown) which removes most types of N-linked carbo-

216

hydrates from glycoproteins [18]. Similarly, the same pattern of radiolabeled proteins was obtained whether membranes were incubated with endoglycosidases for 24 or 96 h or whether a second addition of fresh endoglycosidase F or H or glycopeptidase F was made 17 hours after the first addition and incubation was continued for anoth- er 17 h. These results seem to indicate that N-linked carbohydrates were removed as far as possible by the endoglycosidases under the conditions used.

Thus, in contrast to endoglycosidase D which seemed not to be able to reduce the molecular weight of proteins Psi or P55, a significant reduction of their molecular weight was obtained when membranes were incubated with endoglycosidases H or F. Since the enzyme preparations used did not contain any proteases (as measured by the manufacturer and indicated by the only limited reduction in apparent molecu- lar weight of certain but not all proteins even after prolonged treatment with enzymes), and since incubation was performed in the presence of the protease inhibi- tors PMSF and EDTA (and 1,10-phenanthroline in the case of glycopeptidase F), it seems safe to conclude that any reduction in apparent molecular weight obtained was due to a specific cleavage of asparagine-linked carbohydrates from proteins Psi or P55- Judged by the substrate specificity of the enzymes used, these results suggest the presence of mannose rich, hybrid or complex N-linked carbohydrates on proteins

Psi and P55. Interestingly the same two labeled proteins with apparent molecular weight 48,000

and 51,000 were observed in cerebellum and hippocampus after treatment of mem- branes with endoglycosidase H. The results from cerebellar membranes seem to indi- cate that only part of protein P51 was a substrate for this enzyme. The results from hippocampal membranes and the more intense labeling of the protein with molecular weight 51,000 in hippocampus compared with that in cerebellum seem to indicate that protein P55 is converted to a protein with molecular weight 51,000 by this enzyme.

Similarly, the same three photolabeled proteins (tool. wt. 44,000, 48,000 and 51,000) were observed in both brain regions after treatment of membranes with endoglycosidase F. Whereas in cerebellum only one protein with apparent molecular weight 44,000 was strongly labeled, in hippocampus labeling of all three proteins was about equally strong. This seems to indicate that most of protein P51 was converted to the protein with apparent molecular weight 44,000, whereas protein P55 which is present in cerebellar membranes in only small amounts [11], was converted to two proteins with apparent molecular weights 48,000 and 51,000. Thus, neither protein P51 nor protein P55 seems to be homogeneous in respect to its susceptibility towards endoglycosidases. This might be due to a difference in the carbohydrate structure of part of the proteins [2] (resistance or inaccessibility of some oligosaccharide chains to the endoglycosidases) or to the existence of several proteins with different carbo- hydrate content, comigrating with proteins P51 or P55. Since the apparent molecular weight of proteins Ps~ and P55 each was reduced to a similar extent after treatment with endoglycosidases, from the present data no conclusion can be reached on a pos- sible difference in the carbohydrate content of these proteins.

The present results are in agreement with a previous study [16] indicating that pro-

217

teins photolabeled by [3H]flunitrazepam in membranes from cerebellum, hippocam- pus or cerebral cortex differed in their apparent molecular weight after complete re- moval of their asparagine-linked carbohydrate content. In addition, a reduction in the apparent molecular weight of benzodiazepine receptor subunits similar to that obtained in the present study was observed when purified benzodiazepine receptors were treated with endoglycosidase F [7]. Thus, deglycosylation of purified ~-subunits (apparent mol. wt. 53,000) of the 7-aminobutyric acid (GABA)/ benzodiazepine receptor complex yielded a major protein with apparent molecular weight 44,000- 46,000, as identified by an 0t-subunit-specific polyclonal antibody. Since similar re- suits were obtained after treatment of the purified receptor with trifluoromethane sul- fonic acid which cleaves off carbohydrate peripheral groups both O-linked and N- linked, it was concluded that the completely deglycosylated ct-subunits have an apparent molecular weight of 44,000-46,000. Other experiments indicated that the completely deglycosylated fl-subunits (apparent mol. wt. 57,000) have an apparent molecular weight of 55,000 [7]. This reasonably well fitted with the calculated mole- cular weight of the proposed mature ~t-subunit (429 amino acids, mol. wt. 48,800) and fl-subunit (449 amino acids, mol. wt. 51,400) [8].

Recently, at least two related but distinct genes have been described and sequenced which encode for homologous but different ~-subunits of the GABA/benzodiazepine receptor [9]. In addition, it has been demonstrated that the various proteins specifi- cally photolabeled by [3H]flunitrazepam all are different ~-subunits of the GABA/ benzodiazepine receptor complex [12]. In contrast to hippocampal membranes where at least two different benzodiazepine binding subunits @t-subunits) are enriched, in the purified benzodiazepine receptor preparation used by Mamalaki et al. [7] pre- dominantly only one a-subtype protein was present. Thus, the present results support and extend these previous results by indicating that different benzodiazepine binding proteins (~-subunits) give rise to different deglycosylated proteins. A possible differ- ence in the amino acid structure of these proteins and their possible identity with the proteins derived from the recently described different ct-subunit genes [9] has to be further investigated.

This work was supported by the Fonds zur F6rderung der wissenschaftlichen Forschung in Osterreich. The participation of A. Eichinger and G. Drexler in some of the described experiments is gratefully acknowledged.

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