WO1993025678A1

WO1993025678A1 - BETA-SUBUNIT OF A Na+/D-GLUCOSE COTRANSPORTER

Info

Publication number: WO1993025678A1
Application number: PCT/EP1993/001343
Authority: WO
Inventors: Hermann Koepsell
Original assignee: MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority date: 1992-06-05
Filing date: 1993-05-28
Publication date: 1993-12-23
Also published as: DE4218669C1

Abstract

A protein is capable of regulating the activity of Na+/glucose cotransporters. The cDNA which codes for this protein has been cloned from kidneys. The regulatory activity of the disclosed protein has been shown by co-expression in Xenopus oocytes of the disclosed cDNA with the cDNA which codes for the subunit responsible for transport functions (α-subunit) and by kinetic measurements of the glucose or methyl-α-D-glucopyranoside. This new protein offers for the first time the possibility to pharmacologically influence the transport of glucose by Na+/glucose cotransporters. Also disclosed are the nucleic acid sequences that code for the new protein, as well as a process for producing the same by recombinant DNA technology.

Description

BETA UNIT OF A NA + / D-GLUCOSE COTRANSPORTER

description

The present invention relates to a protein with the biological activity of a / 3 subunit of a Na ⁺ / glucose cotransporter, and to a nucleic acid fragment which contains a nucleotide sequence which codes for the same protein. The invention further relates to a recombinant vector which contains the nucleic acid fragment and to a host cell which contains this vector. The invention further relates to a method for the production of the protein and a pharmaceutical composition containing this protein.

Na ⁺ / glucose cotransporters occur in mammals, for example in the kidney and intestine. In the intestine, the transport protein is responsible for the glucose uptake from the diet. In the kidney, it transports the filtered glucose back into the blood. Because of the different food supply in the intestine and the different glucose concentration in the different sections of the kidney tubule, the activity of the Na ⁺ / glucose cotransport protein has to be regulated.

It has been shown that the Na ^ / glucose cotransporter can effectively transport glucose at both very low and very high glucose concentrations.

The transport properties of the transport protein can be regulated specifically within different kidney sections according to the biological requirements. An overview of the transport properties of various glucose transporters can be found in Annu. Rev. Biochem. 60, p.757 ff, 1991. By Ikeda et al. Starting from RNA isolated from rabbit intestine, a gene was cloned which codes for a protein which is able to transport Na ⁺ together with glucose (Ikeda TS et al., Membrane Biol. 110, 87-95, 1989) . Furthermore, the gene for a human protein with Na / glucose cotransport properties was cloned and designated SGLT1. The rabbit and human clones have 85% homology at the protein level. The proteins encoded by these genes carry out a Na-dependent glucose transport, which however cannot be regulated. If the genes are expressed in Xenopus oocytes (Ikeda TS et al., Membrane Biol. 110, 87-95, 1989) or cell culture cells (Birnir, B. et al., Biochim. Biophys. Acta 1048, 100-104, 1990) , one finds other transport properties than under in vivo conditions. Obviously, the known non-regulatable proteins represent only a part of the complete regulable Na ⁺ / glucose cotransporter. They are referred to below as “subunits”.

It would be a major medical advance if the activity of the Na / glucose cotransporter could be influenced in a targeted manner. Inhibiting the Na ⁺ / glucose co-transporter in the intestine would prevent or drastically reduce glucose uptake. This would be desirable for diabetics, for example, since it would reduce the need for insulin and allow the diabetic to relax the dietary requirements.

Inhibition of the Na / glucose cotransporter in the kidney could also be used in diabetics to improve the regulation of blood glucose levels achieved during insulin therapy. That would be one of the reasons important because the late damage that occurs in diabetes can often be attributed to hitherto unavoidable, intermediate increases in the blood sugar level. If the Na ⁺ / glucose cotransporter were inhibited in the kidney, there would be an increased glucose excretion in the urine and thus a decrease in the glucose concentration in the blood.

Inhibition of the Na / glucose cotransporter in the intestine and kidney would furthermore lead to reduced energy absorption in the intestine and to a considerable loss of energy in the kidney. The regulation of the transport performance of the Na ⁺ / glucose cotransporter could thus also be used in the therapy of obesity.

The object of the present invention is to provide a protein which enables regulation of the activity of Na / glucose cotransporters.

This object is achieved according to the invention by a protein with the biological activity of a / 3 subunit of a Na / glucose cotransporter which is encoded by a nucleotide sequence selected from a DNA sequence which codes for the amino acid sequence of FIG. 6, and selected from nucleic acid sequences that hybridize with this sequence and code for a protein with the biological activity of the jS subunit of a Na / glucose cotransporter.

Another object is to provide a nucleic acid fragment which codes for a protein of the type mentioned at the beginning. This object is achieved according to the invention by a nucleic acid fragment containing a nucleotide sequence selected from a DNA sequence comprising the sequence from nucleotide position 1 to 4529 of FIG. 6 and selected from nucleic acid sequences, preferably DNA sequences which correspond to the Hybridize sequence from FIG. 6 and code for a protein with the biological activity of the β subunit of a Na ⁺ / glucose cotransporter.

Another object is to provide a recombinant vector containing the above-mentioned nucleic acid fragment.

This object is achieved according to the invention by a vector according to claim 9.

Another object is to provide a host cell which contains the vector mentioned above.

This object is achieved according to the invention by a host cell according to claim 13.

Another object is to provide a pharmaceutical composition containing the above protein.

Another object is to provide a method for producing the protein mentioned in the introduction.

This object is achieved according to the invention by a method comprising cultivating a host cell containing the recombinant vector mentioned above and isolating the expressed protein. ^{■ •} The invention is explained in more detail below with reference to FIGS. 1 to 8.

Figure 1: In vitro expression of a single polypeptide by plasmid P20, the polypeptide specifically reacting with the onoclonal antibody R4A6. Reticulocyte lysate with (c, d) or without L- [35S] methι.onιn (a, b, e, f, g) was mixed with water (P20 -) or with P20 cRNA

(P20 +), incubated at 37 ° C., and the samples were used for SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The gel was dried and autoradiographed (c, d), or the proteins were transferred to nitrocellulose and either stained with amido black (a, b) or the monoclonal antibody R4A6 (e, f) or an IgM antibody as a control ( g) exposed.

Figure 2: Different effects of P20 on Na ⁺ / glucose cotransport in Xenopus oocytes with high and low endogenous transport activity. Xenopus laevis oocytes with low activity of Na-dependent D-glucose transport or methyl-α-D-glucopyranoside (AMG) transport (a, b) and those with high endogenous transport activity (c, d) were identified at 50 μl water (c) or with 50 μl water containing 0.3 ng P20-CRNA, (P20) injected. After 48 h (18 ° C.) incubation, the uptake of 50 μM D-glucose and 50 μM AMG was measured in the presence or absence of Na. A representative experiment is shown. Na-dependent uptake rates of 10 oocytes are the mean +/- se. (Standard deviation / n, where n = number of individual measurements). Figure 3: Dependency of the AMG transport, expressed in Xenopus oocytes, on the amount of SGLTl cRNA (a) and P20 cRNA injected, which was co-injected with SGLTl cRNA (b, c). In (a) different amounts of SGLTl cRNA were injected, in b) 0.15 ng SGLTl cRNA per oocyte was injected together with different amounts of P20 cRNA, and in c) 1.2 ng SGLTl cRNA and different amounts were injected P20-CRNA injected.

FIG. 4: Comparison of the glucose dependence of the Na / glucose cotransport after expression of SGLTl cRNA or of stoichiometric amounts of SGLTl cRNA and P20-CRNA.

Different amounts of SGLTl cRNA were injected per oocyte alone (a, 0.145 ng; b, 0.5 ng; c, 1.2 ng) or together with a stoichiometric amount of P20 cRNA (a, 0.51 ng; b , 1.75 ng; c, 4.2 ng). In the presence of Na and various AMG concentrations, the AMG uptake in oocytes injected with cRNA and with water was measured and the expressed uptake rates were calculated (mean +/- sem of 8 to 10 oocytes are shown). The straight lines were adjusted on the assumption that there is 1 transport point, while the curves were calculated on the assumption that there are 2 glucose transport points. Open symbols represent values that were obtained after injection of SGLTl cRNA alone. V in pmol x mg xh ^-1 : (a insertion) 15.5 +/- 0.35, (b) 176.9 +/- 2.2, (c) 570.2 +/- 10.5; K _{Q 5} in μM: (a insertion) 117 +/- 6; (b) 122 +/- 3; (c) 126 +/- 5. Closed symbols represent values which were obtained after co-injection of SGLTl cRNA and P20 cRNA. V (high affinity) in pMol x Oocyte ^"1 xh ^-1 : (a) 116.0 +/- 2.5; (b) 56.5 +/- 1.0; (c) 66.5 +/- 3.5; V _m (low Affinity) in pMol x oocyte ^-1 xh ^"1 : (a) 454.0 +/- 1.0; (b) 513.5 +/- 1.5; (c) 502.0 +/- 44.0; K _{Q 5} (high affinity) in µM: (a) 28.5 +/- 0.6; (b) 16.7 +/- 1.1; (c) 21.4 +/- 3.0; K _{Q 5} (low affinity) in M: (a) 1.88 +/- 0.08; (b) 0.91 +/- 0.03; (c) 1.37 +/- 0.14.

Figure 5: Comparison of the _. Glucose dependence of the Na ⁺ / glucose cotransport after expression of SGLTl cRNA or of SGLTl cRNA with an excess of P20 cRNA. 0.3 ng'SGLTl cRNA (o) or 0.3 ng SGLTl cRNA + 8.5 ng P20-CRNA (.) Were injected per oocyte. The expressed uptake rates were measured and shown as in FIG. 4. Injection of SGLT1: V = 110 +/- 6 pmol x oocyte ^-1 xh ^-1 , K ^ = 96 +/- 5 μM; Injection of SGLT1 + P20: V _maχ = 124 +/- 7 pmol x oocyte ^-1 xh ^-1 ; K _m = 92 +/- 5 µM.

FIG. 6: nucleotide sequence and putative amino acid sequence of P20. A possible translation start site (nucleotides 118 to 127) and two consensus polyadenylation signals (nucleotides 1861-1866, 2922-2927) are underlined. 15 amino acids are framed at the C-terminus and can form a membrane-spanning α-helix. Six potential N-glycosylation sites are indicated by circles.

Figure 7: The figure shows frozen sections through the animal pole of oocytes of the clawed frog Xenopus laevis, which were developed with a monoclonal antibody directed against the ^ subunit (R4A6). The antibody was marked with a fluorescent substance and then detected in a fluorescence microscope. a) shows an oocyte after injection of cRNA which codes for the α subunit. Here you can see a weak antibody reaction in the plasma membrane. This indicates that the oocyte has an endogenous β-subunit (P20 protein), which is incorporated into the membrane together with the additionally expressed α-subunit.

b) shows an oocyte after co-injection of cRNA of the α subunit (SGLT1) and cRNA of the β subunit (P20). Here the antibody directed against the β-subunit reacts clearly with the plasma membrane.

c) shows a control oocyte that was injected with water. The antibody does not react here.

Figure 8: The figure shows an experiment in which it is demonstrated that the regulatory / 3 subunit of the Na ⁺ / D-glucose cotransporter also occurs in the small intestine. Polymerase chain reactions were carried out with oligonucleotide primers derived from the sequence of the P20 clone, which correspond to positions 793-811 (5 'GCTTTTG-GTCATTAGTAGT 3') and 1562-1578 (3 'GGATGTATTGGTACCTT 5', opposite strand) in FIG. 6 carried out mRNA from the pig kidney (b), on mRNA from the pig intestine (c), on mRNA from the pig muscle (d) and on a control without mRNA (e); (a) shows the molecular weight marker with fragment size of 1150 bp, 1000 bp, 680 bp and 490 bp as the lower 4 bands. The left part of the illustration (A) shows an agarose gel stained with ethidium bromide, on which the resulting reaction products were separated. The right part of the illustration (B) shows a parallel experiment in which the products separated in the agarose gel were transferred to nylon foil and then with one of the sequence of the P20 derived, but in this case radioactively marked oligonucleotide were hybridized. (The radioactively labeled oligonucleotide came from the region between the two oligonucleotide primers used for the PCR reaction). The experiment shows that transcripts of the expected length (786 bp) are produced from the pig kidney and pig intestine using the mRNA. It proves that there is a protein in the pig intestine that is very similar to or identical to the β-subunit cloned from the kidney.

To isolate the protein according to the invention, a monoclonal antibody was used which is directed against the Na / glucose cotransporter from porcine kidney, but does not react with the already known SGLT1 protein. This antibody (R4A6) stimulates Na-dependent phlorizin binding in pig kidney and rat intestine (Haase, W. et al., Eur. J. Cell Biol. 52, 297-3009, 1990; Koepsell, H. et al., J Biol. Chem. 263, 18419-18429, 1988). In rats, pigs, rabbits and humans it binds to a polypeptide with a molecular weight of approximately 75,000, which is located in the brush border membrane of proximal kidney tubules. The antigenic polypeptide of R4A6 shows a similar immunohistochemical distribution as other antibodies which are directed against the Na ⁺ / glucose transporter (Haase, W. & Koepsell, H., Eur. J. Cell Biol 48, 360-374, 1989; Takata, K. et al., Histoche. Cytochem. 39, 287-298, 1991).

With the above-mentioned monoclonal antibody R4A6, a cDNA expression bank was screened which had been prepared from RNA from porcine kidney cortex. A positive clone (P20) with a 4.5 kb length cDNA insert was isolated. P20 does not hybridize with SGLTl cDNA and with cDNAs encoding Na ⁺ independent glucose transporters (James, DE et al., Nature 338, 83-87, 1989; Celenza, JL et al., Proc. Natn. Acad. Sei USA 85, 2130-2134, 1988; Mueckler, M. & Lodish, HF, Cell 44, 629-637, 1986).

P20 was translated in vitro using a customary reticulocyte lysate in order to estimate the size of the polypeptide encoded by P20 and to test the reactivity against control antibodies. After adding P20 cRNA to the reticulocyte lysate, a single polypeptide was synthesized, which runs in the SDS polyacrylic gel with an apparent molecular weight of 70,000 and reacts specifically with the monoclonal antibody R4A6 (FIG. 1).

P20-CRNA was injected into Xenopus laevis oocytes in order to determine the effect of the polypeptide encoded by P20 on the endogenous Na / glucose cotransport. Consistent with previous observations (Weber, W.-M. et al., Membrane Biol. 111, 93-102, 1989), the endogenous Na ⁺ / glucose cotransport varies considerably depending on the various oocyte preparations. Consequently, the uptake rates of 50 μM D-glucose or methyl-α-D-glucopyranoside (AMG), which depend on the Na gradient, varied between 0.5 +/- 0.1 and 0.4 +/- 0.05 pMol x oocyte ^-1 xh ^-1 and 38 +/- 4 or 1.9 +/- 0.2 pMol x oocyte ^-1 xh ^-1 . After injection of 0.1 to 0.5 ng per oocyte P20 cRNA was then increases dependent on the Na ⁺ gradient uptake of 50 .mu.M D-glucose or AMG when dependent on endogenous Na ⁺ gradient D-Glucoseaufnähme (50 .mu.M ) of oocytes injected with water was less than 1.5 pmol x oocyte-xh. However, the uptake rate was reduced when the endogenous uptake was greater than 9 pmol x oocyte-x (Figure 2). These effects were also observed when injected with cRNA encoded only by nucleotides 1 to 1826 of P20 (Figure 6). No effects on endogenous Na ⁺ / glucose cotransport were observed when anti-sense cRNA was injected from P20 or when the oocytes were incubated for only 3 h after injection of sense cRNA. To test the specificity, the effects of P20 cRNA on endogenous Na ⁺ -L-glutamate cotransport (Steffgen, J. et al., Biochim. Biophys. Acta 1066, 14-20, 1991) and on Na ⁺ / K ⁺ pump (Schmalzing, G. et al., W. Biochem. J. 297, 329-336, 1991). Neither the uptake of L-glutamate depending on the Na ⁺ gradient (measured at different

Glutamate concentrations) or the ouabain binding to the (Na / K) - ATPase or the Rb ⁺ uptake which can be inhibited by ouabain by the (Na ⁺ / K ⁺ ) - ATPase was changed.

It was also investigated whether the expression of the Na ⁺ / glucose cotransport was changed by injecting SGLTl cRNA into Xenopus oocytes by injecting P20 cRNA at the same time. Injection of SGLTl cRNA into oocytes resulted in a dramatic stimulation of AMG and D-glucose uptake in the presence of 110 mM Na ⁺ . The uptake rates were reduced by more than 95% and 98% respectively when Na ^{+ was} replaced by tetramethylammonium or when 100 μM phlorizin was added.

FIG. 3a shows the correlation between AMG transport and the amount of SGLT1 cRNA injected. Co-injections of P20-CRNA with SGLTl-cRNA, which were carried out with many different oocyte preparations, showed that the effects of P20 depended on the total amount of SGLTl-cRNA injected and on the ratio between the Amount of SGLTl cRNA and P20 cRNA injected. P20 cRNA stimulated the transport of 50 μM AMG between 2 to 25 times when 0.2 ng or less SGLTl cRNA was injected per oocyte (10 experiments, molar ratios of P20-CRNA to SGLTl cRNA from 1.8 to 4.5). When the amount of SGLTl cRNA and P20-CRNA injected per oocyte was between 0.7 and 1.5 ng, co-injection of P20-CRNA inhibited the transport of 50 μM AMG between 39% and 87% (sem 7%) ( 15 experiments, molar ratios of P20-CRNA to SGLTl cRNA from 1.6 to 3.1). When measuring the uptake at different molar ratios between the injected SGLTl cRNA and P20 cRNA, maximum stimulation and inhibition of the AMG uptake was found with approximately the same stoichiometric ratio between the cRNAs (FIG. 3b, c). This is a clear indication that the observed, stimulating and inhibiting effects result from a specific interaction of SGLT1 and P20, whereby it can be assumed that this interaction takes place at the protein level since both clones have no nucleotide sequence homology.

In order to determine whether the interaction between SGLT1 and P20 takes place in the cytoplasm a and / or whether the P20 protein and SGLTl protein interact in the plasma membrane, the functional properties of the Na ⁺ / glucose cotransport were compared, that by SGLT1 and is expressed by SGLT1 + P20.

FIG. 4 shows the glucose dependency of the AMG uptake after injection of three different amounts of SGLTl cRNA alone and after co-injection of SGLTl cRNA with a fixed stoichiometric amount of P20-CRNA. After injection Different amounts of SGLTl cRNA were used to obtain Michaelis-Menten kinetics with IC values between 96 +/- 5 and 126 +/- 5 μM. With increasing amounts of injected SGLT1 cRNA, _{χ of} the transport increased, whereas the K_ value remained constant. This indicates that the properties of the transporter expressed by SGLT1 are independent of the transporter density in the membrane. The co-injection of a fixed stoichiometric amount of P20-CRNA and SGLTl-cRNA has two effects: 1. An increase in the number of functional transporters (increase in _rnaχ ) when low amounts of SGLTl-cRNA were injected, which leads to a maximum expression (FIG. 4a, b). 2. A functional change in the transporter in the membrane, which indicates the generation of transport sites with high and low affinity (FIGS. 4a, b, c).

Consequently, with different amounts of a stoichiometric mixture of SGLTl cRNA and P20-CRNA, AMG transport sites with high and low affinity were obtained with K _{Q 5} values of 17 to 29 μM and 0.9 to 1.9 mM (FIG. 4a , b, c). Expression of one type of glucose binding site by SGLT1 and two types of glucose binding site by SGLT1 +/- P20 was shown by interaction of the competitive inhibitor phlorizin with glucose binding sites on the extracellular side of the transporter (Koepsell, H. et al., Membrane Biol. 114, 113-132, 1990). After injection of SGLTl cRNA, a site for the inhibition of AMG transport by phlorizin was observed (K- = 5.0 +/- 0.1 μM), whereas inhibition sites of high and low affinity (K ^ (high affinity) = 0.27 +/- 0.01 μM, K ^ (low affinity) = 29 +/- 6 μM) after injection of a fixed stoichiometric amount of SGLTl cRNA and P20 cRNA. These values show that the SGLTl protein and the P20 protein interact in the membrane. The theoretical possibility that P20 changes the function of SGLT1 indirectly via the membrane potential or via the Na concentration in the oocytes can be excluded by the fact that the membrane potential and the intracellular Na ⁺ concentration in the presence of glucose do not by P20-CRNA was changed. The observation that P20 cRNA does not alter the AMG uptake expressed by SGLT1 when an excess of P20 CRNA is co-injected (Figure 3b, c) is an indication that two P20 polypeptides are likely to contain the SGLT1 protein ( interact with a dimer), and that the effects described above on the transporter concentration in the membrane and on the function of the transporter are caused by the interaction of a P20 polypeptide on the SGLTl dimer.

The experiment in Figure 5 is consistent with this hypothesis. No significant stimulation of V was observed after co-injection of SGLTl cRNA and an excess of P20 cRNA, and the glucose dependency was approximately the same as after injection of SGLTl cRNA alone.

Furthermore, it was possible in the context of the invention to demonstrate that the β-subunit encoded by clone P20 is involved in the dietary regulation of the Na / glucose co-transporter:

Using RNA samples isolated from the small intestine of a) two-week-old sheep during lactation, b) adult sheep (2 years old) and c) adult sheep infused with D-glucose. Ren, polymerase chain reactions were carried out. The primer pairs described in the legend to FIG. 8 were used as oligonucleotide primers. It was found that only in the young sheep (group a) and in the adult sheep infused with glucose (group c) did reaction products occur with the expected length.

Since Shirazi-Beechey et al. (Journal of Physiology 437, 699-708, 1991), it could be shown that the Na ⁺ -D-glucose cotransport in the intestine of sheep is regulated by the food supplied, the result achieved means that the amount of P20- mRNA correlated with the activity of the Na ⁺ / glucose co-transporter under in vivo conditions.

The nucleotide sequence and the amino acid sequence of P20 derived therefrom is shown in FIG. 6. The open reading frame (ORF) begins at the EcoRI cloning site at nucleotide position 1 and is identical to the open reading frame of a β-galactosidase fusion protein in λ Zap phage (Short, JM et al., Nucleic Acids Res. 16 , 7583-7600, 1988). A protein with 522 amino acids with a molecular mass of 56245 Da is derived from this open reading frame. The open reading frame may extend beyond the 5 'end of P20, ie P20 may not contain the nucleotides coding for the N-terminal amino acids of the native protein. In reticulocyte lysates and Xenopus oocytes, protein synthesis probably begins at the first ATG at nucleotide position 125, because a computer analysis showed that nucleotides 118 to 127 can serve as a translation initiation sequence in eukaryotes (Staden, R., Nucleic Acids Res. 12, 505-519; Kozak, M., Nucleic Acids Res. 12, 857-872, 1984). The 3 'non-coding region of P20 is 2962 bp long and contains 2 consensus polyadenylation signals (AATAAA). A comparison of the nucleic acid sequence of P20 with databases shows no homology with other sequences. An analysis of the hydrophobicity (Kyte, J. & Doo-little, RF, J. Molec. Biol. 157, 105-132, 1982) and the secondary structure (Garnier, J. et al., J. Molec. Biol. 120, 97-120, 1978) showed that the P20 protein is relatively hydrophilic and presumably has three larger α-helical regions (amino acids 2 to 21, 247 to 269 and 506 to 520). According to Rao and Argos (Rao, JKM & Argos, P., Biochim. Biophys. Acta 869, 197-214, 1986), the carboxy-terminal α-helix could be membrane-spanning.

The P20 protein contains 24% charged amino acids which are arranged in clusters and which are concentrated at the N-terminus. Furthermore, 6 N-linked glycosylation sites are predictable (Bause, E., Biochem. J. 209, 331-336, 1983). All indications suggest that the major part of the protein is on the outside of the membrane. This interpretation agrees with the observation that the antibody R4A6 binds to the extracellular part of the Na / glucose cotransporter. The data suggest that the complete Na ⁺ / glucose cotransporter is a heterooligomer consisting of SGLT1 and P20 type polypeptides. SGLT1-type polypeptides (α-subunits) represent the membrane-spanning pump, but the physiological properties of the transporter with transport points with low and high affinity only after the composition with the P20-type polypeptides (β- Subunits) can be obtained. By identifying and isolating the β subunit of the Na ⁺ / glucose cotransporter and elucidating its primary structure, it is possible for the first time to influence the activity of the Na ⁺ / glucose cotransporter in the kidney and intestine. While the .alpha.-subunit is an integral membrane protein which only protrudes from the membrane with short sections at a few points, the .beta.-subunit is a protein which is anchored to the cell membrane at one end and closed more than 90% protrudes from the membrane into the intestinal lumen or into the lumen of the proximal renal tubule. This extracellular part can be influenced by pharmaceuticals. Since the β-subunit regulates the activity of the Na ⁺ / glucose co-transporter, this transport activity can be influenced by pharmaceuticals via the β-subunit. The β-subunit can serve as a receptor for various types of pharmaceutical active substances, the active substances being able to bind easily to the accessible β-subunit and thus being able to modulate the activity of the transport, as is possible, for example, with phlorizin.

In addition to the pharmacological influence on the transport activity by active substances which bind to the transport protein, a genetic influence is also possible. In this way, a targeted inhibition of the mRNA synthesis of this protein can be achieved pharmacologically via the regulatory sequences of the gene for the β subunit. As a result, less β-subunit is synthesized, and this leads to a greatly reduced incorporation of functional Na ⁺ / glucose cotransport proteins.

In the context of the present invention, a protein with “the biological activity of a β-subunit” is understood to mean a protein or polypeptide which can regulate the activity of a Na ⁺ / glucose cotransporter depending on the glucose concentration. A pharmaceutical composition containing the protein according to the invention can be produced by the customary production process and comprises the customary auxiliaries and carriers.

As mentioned above, the nucleic acid fragments isolated from rabbits and humans, which code for the α subunit of the Na / glucose cotransporter, have a high sequence homology. This also applies to the sequence contained in P20 and other mammalian sequences (see FIG. 8). It is therefore possible, using the cDNA described herein and by means of the methods used in the field of recombinant DNA technology, as described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, New York, 1989) to isolate further DNA sequences from other mammalian species, such as humans, which are necessary for the β subunit of Na ⁺ / Encode glucose cotransporters.

The fact that the regulatory β-subunit of the Na / glucose cotransporter cloned from the pig also occurs in the kidneys and intestines of humans could be demonstrated with the aid of the polymerase chain reaction in the context of the present invention:

Starting from total RNA, which had been isolated from human intestine and human kidney, cDNA was first produced. The synthesized cDNA was then used in the polymerase chain reaction, which was carried out with the following oligonucleotide primers:

1) 5 'CTTAATTCAGCAGGCGG 3' (P20 positions 491-507) and

2) 3 'GGAACAGTAGGTCGAAG 5' (P20 positions 1255-1271, opposite strand). The reaction was carried out as described in Example 5. The reaction batches were then electrophoresed on agarose gels and stained with ethiclumin bromide. A comparison with marker DNA fragments showed that reaction products with the expected length (781 bp) were formed. This result showed that the protein according to the invention also occurs in the intestine and kidney of humans.

The expression of the DNA sequences and the isolation of the expressed proteins of other mammalian species can also be carried out according to the usual methods, such as, for example, in the laboratory manual by Sambrook et al. described.

The nucleic acids coding for the β-subunit can be of natural origin, such as genomic DNAs or cDNAs, also of a synthetic nature, such as DNA that can be produced with the aid of automatic synthesizers, as well as of a semi-synthetic nature. The selection of the vector for taking up the nucleic acid coding for the protein according to the invention depends on the host cell used and is within the range of the person skilled in the art. Suitable vectors are described, for example, in the laboratory manual by Sambrook et al. listed.

The recombinant vectors which carry the nucleic acid fragments according to the invention can be introduced into the corresponding host cells by customary methods, as described, for example, in Sambrook et al., See above. For example, come as host cells Microorganisms in question, such as bacteria of the species E.coli and B.subtilis. Eukaryotic organisms such as yeasts can also be used. It is also possible to use eukaryotic cell culture cells or cultured insect cells. After culturing the host cells which contain the nucleic acid fragment according to the invention, the recombinant proteins with the biological activity of a β-subunit of a Na / glucose cotransporter can be isolated by conventional methods using conventional methods. The protein according to the invention can be used for pharmacologically influencing the Na ⁺ / glucose cotransport activity. So the ß-subunit of the invention

Na ⁺ / glucose cotransporters are assembled together with the α subunit to form an intact complex, purified and crystallized in order to determine the tertiary structure of the transporter by X-ray structure studies. Based on this data, specific active substances that bind to transporters can be produced. The protein according to the invention thus enables for the first time the targeted production of a ligand that binds to the Na / glucose cotransporter. Such a ligand can be used to regulate the activity of a Na / glucose cotransporter, for example to inhibit the activity in diabetics.

The following examples illustrate the invention.

Example 1 In vitro expression of a single polypeptide by P20 which specifically reacts with the monoclonal antibody R4A6. To prepare sense cRNA, purified Bluescript plasmids (available from the Stragene company) containing P20 were linearized with Xhol and the cRNA provided with 5'7meGppp5'G was purified by T3 polymerase synthesized using the mCAP mRNA capping kit from Stratagene. The cRNA was dissolved in diethyl pyrocarbonate (DEPC) treated water and the cRNA concentration was determined (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, New York, 1989 ). 45 μl were used for in vitro translation

Rabbit reticulocyte lysate (Amersham) with or without L- [ ³⁵ S] methionine (15 μCi)) mixed with 5 μl water without or with 0.9 μg P20-CRNA. After 1 h (37 ° C.) incubation, 50 μl 125 mM Tris-HCl pH 6.8, 4% (w / v) SDS, 10% (w / v) β-mercaptoethanol, 20% (w / v ) Added polyethylene glycol and 20 µl aliquots were used for SDS polyacrylamide gel electrophoresis. Western blotting and the immune response with 2 ug / ml mouse IgM antibody or with 2 ug / ml mouse myeloma IgM was performed as described in Koepsell, H. et al. (J. Biol. Chem. 263, 18419-18429, 1988).

Example 2

Different effects of P20 on the

Na ⁺ / glucose cotransport in Xenopus oocytes with low and high endogenous transport activity.

Adult oocytes were collected after collagenase treatment (Schmalzing, G. et al., Biochem. J. 279, 329-336, 1991) and 50 nl of DEPC-treated water without or with cRNA were injected per oocyte. For translation, the oocytes were kept in 5 mM Hepes-Tris pH 7.8, 110 mM NaCl, 3 mM KC1, 2 mM CaCl ₂ , 1 mM MgCl ₂ (ORi) buffer, containing 50 mg / 48 h (18 ° C). 1 gentamicin incubated. The glucose uptake was measured by incubating the oocytes at 22 ° C. with ORi buffer or with 5 mM Hepes-Tris pH 7.6, 110 mM tetramethylammonium, 3 mM KC1, 2 mM CaCl ₂ , 1 mM MgCl ₂ (HT buffer), containing D- [ ¹⁴ C] glucose or D- [C] AMG. Since glucose uptake was always linear for 2 hours, initial uptake rates were routinely measured after 1 hour of incubation. The incubation was stopped by ice-cold ORi or HT buffer and the radioactivity of individual oocytes was measured after their dissolution in 5% (w / v) SDS.

Example 3

Dependence of the AMG transport expressed in Xenopus oocytes on the amount of SGLTl cRNA injected and on P20-CRNA, which was co-injected with SGLTl cRNA.

The transport of 50 μM AMG in Xenopus laevis oocytes was measured in the presence of 110 mM Na (see Example 2) and corrected for the AMG uptake measured in oocytes injected with water.

Example 4

Nucleotide sequence and derived amino acid sequence of P20. MRNA purified from pig kidney cortex (Oberleithner, H. et al., Methods Enzymol. 191, 437-449, 1990) was used to produce a cDNA library in λ-Zap phage (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, New York, 1989) using oligo-dT. The library was screened for antibodies using the monoclonal antibody R4A6 using a second antibody coupled to phosphatase (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, New York , 1989). A positive one Plaque was isolated and the pBluescript SK plasmid (with cDNA insert) was excised using helper phage R408. XL1-Blue cells were transfected and the plasmid DNA was purified from liquid cultures (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, New York, 1989). Using the dideoxynucleotide chain termination method (Korneluk, RG et al., Gene 40, 317-323, 1985), both strands of the double-stranded plasmid DNA were sequenced.

Example 5

Polymerase chain reaction for the detection of cDNA, which is synthesized starting from RNA isolated from human intestine and human kidney and is homologous to the P20 cDNA.

The PCR reaction was carried out in a reaction frame of 50 μl in the presence of human RNA; 67 mM Tris HC1 pH 8.8; 6.7 mM MgCl ₂ ; 1 mM each of dNTPs; 0.17 mg / ml bovine serum albumin; each carried out 20 pmol primer.

Procedure: 5 minutes denaturation at 94 ° C, addition of 2.5 U tag polymerase (hot start) 5 minutes annealing at 45 ° C, 40 minutes reaction at 72 ° C (for the synthesis of the DNA strand); then 35 cycles of 1 minute each at 94 ° C, 1.5 minutes at 48 ° C, 3 minutes at 72 ° C.

Claims

1. Nucleic acid fragment containing a nucleotide sequence which codes for a protein with the biological activity of a β subunit of a Na ⁺ / glucose co-transporter, the nucleotide sequence being selected from a DNA sequence comprising a sequence which is for the amino acid sequence 6, and from nucleic acid sequences which hybridize with this sequence and code for a protein with the biological activity of the β subunit of a Na / glucose co-transporter.

2. Nucleic acid fragment according to claim 1, characterized in that the nucleotide sequence codes for an amino acid sequence which comprises the amino acid sequence shown in FIG. 6 from amino acid position 42 to 522.

3. Nucleic acid fragment according to claim 1, characterized in that it comprises the DNA sequence from position 125 to 1568 from FIG. 6 or a sequence which hybridizes to it.

4. Nucleic acid fragment according to claim 1, characterized in that it comprises the DNA sequence from position 1 to 1568 from FIG. 6 or a sequence which hybridizes to it.

5. Nucleic acid fragment according to claim 1, characterized in that it is of synthetic, semi-synthetic or natural origin.

6. Nucleic acid fragment according to claim 1, characterized in that the nucleotide sequence codes for a β-subunit of a mammalian Na / glucose cotransporter.

7. Nucleic acid fragment according to claim 6, characterized in that the nucleotide sequence codes for a β-subunit of a porcine Na ⁺ / glucose cotransporter.

8. Nucleic acid fragment according to claim 6, characterized in that the nucleotide sequence for a β-subunit of a human

Coded Na / glucose cotransporters.

9. Recombinant vector containing a nucleic acid fragment according to one of claims 1 to 8.

10. Recombinant vector according to claim 9, characterized in that it is an expression vector.

11. Recombinant vector according to claim 10, characterized in that it is an expression vector for prokaryotes.

12. Recombinant vector according to claim 10, characterized in that it is an expression vector for eukaryotes.

13. Host cell containing a vector according to one of claims 9 to 12.

14. Host cell according to claim 13, characterized in that it expresses the protein with the biological activity of a β-subunit of a Na / glucose co-transporter.

15. Protein with the biological activity of a β subunit of a Na / glucose cotransporter, which is encoded by a nucleic acid fragment according to one of claims 1 to 8.

16. Protein according to claim 15, characterized in that it comprises the amino acid sequence from amino acid position 42 to 522 in Fig. 6.

17. Protein according to claim 15, characterized in that it comprises the amino acid sequence from amino acid position 1 to 522 in Fig. 6.

18. A method for producing a protein having the biological activity of a β subunit of a Na ⁺ / glucose cotransporter, comprising the steps of culturing a host cell according to claim 13 and isolating the expressed protein.

19. A pharmaceutical composition containing a protein according to any one of claims 15 to 17.

20. Use of the protein according to any one of claims 15 to 17 as a receptor for an active pharmaceutical ingredient.

21. Use of the protein according to any one of claims 15 to 17 for the preparation of a ligand binding to a Na ⁺ / glucose cotransporter.

22. Use according to claim 21, characterized in that the ligand is an inhibitor of the Na ⁺ / glucose cotransporter.