WO1991008232A1 - Clonage et expression de la proteine ht7 specifique de la barriere hematoencephalique - Google Patents

Clonage et expression de la proteine ht7 specifique de la barriere hematoencephalique Download PDF

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Publication number
WO1991008232A1
WO1991008232A1 PCT/EP1990/002000 EP9002000W WO9108232A1 WO 1991008232 A1 WO1991008232 A1 WO 1991008232A1 EP 9002000 W EP9002000 W EP 9002000W WO 9108232 A1 WO9108232 A1 WO 9108232A1
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protein
amino acid
brain
antigen
blood
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PCT/EP1990/002000
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German (de)
English (en)
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Harald Seulberger
Friedrich Lottspeich
Werner Risau
Sigrid Henke-Fahle
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Publication of WO1991008232A1 publication Critical patent/WO1991008232A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the unique properties of endothelial cells in the brain, which form the blood-brain barrier, are expressed by the expression of specific cell surface molecules, such as the HT7 protein. Purification, cloning and expression of this molecule showed that it is a new transmembrane glycoprotein with two Im unglobulin-like domains. This protein can be a receptor that participates in the cell surface recognition at the blood-brain barrier.
  • Impermeable tight junctions zonula occludens
  • specific transport systems in endothelial cells of the brain capillaries are responsible for the blood-brain barrier in higher vertebrates (TS Reese & MJ Karnovsky, J. Cell Biol. 34, 207-217 (1967) and AL Betz & GW Goldstein, Annu. Rev. Physiol. 48, 241-250 (1986)).
  • astrocytes the end feet of which lie tightly on the discharge side of CNS capillaries, induce blood-brain barrier properties in endothelial cells in vivo (RC Jan-er and MC Raff, Nature 325, 253-257 (1987) ).
  • Two processes are involved in the formation of a functional vascular system of the blood-brain barrier.
  • angiogenesis the formation of capillaries from the existing perineural vascular system, causes the migration and proliferation of endothelial cells. This process is presumably regulated by soluble brain-derived factors (W. Risau, Proc. Natl. Acad. Sei. USA 83, 3855-3859 (1986)).
  • the blood vessels thus formed differentiate into barrier capillaries.
  • astrocytes play a major role in the latter process, the mechanisms remain unclear (RC Janzer and MC Raff, Nature 325, 253-257 (1987)). This is mainly due to the lack of an in vitro model system. As a result, markers that specifically recognize in vitro endothelial cells of the blood-brain barrier are valuable for the development of such a system.
  • the blood-brain barrier-specific HT7 antigen can be characterized using antibodies (W. Risau, R. Hallmann, U. Albrecht & S. Henke-Fahle, EMBO J. 5, 3179-3183 (1986)). Monoclonal antibodies to HT7 are available from mice immunized with the nasal retina of 7-day-old chickens according to the protocol of Mattew and Patterson (Cold Spring Harbor, Symp. Quant. Biol. 48. (1983), 625-631) with Cyclophos - Pha id were immunosuppressed and in which the anti-body formation was stimulated by the administration of sleeping retina. The mice treated in this way contain polyclonal antibodies.
  • a monoclonal antibody against HT7 can be produced in the usual way by fusing NS1 myeloma cells with spleen cells of these mice. The specificity of the antibodies obtained can be tested on frozen sections from the retina and tectum. It was found that the expression of the cell surface marker induced HT7 in endothelial cells which had penetrated into transplanted brain tissue (W. Risau, R. Hallmann, U. Albrecht & S. Henke-Fahle, EMBO J. 5, 3179-3183 (1986)). It is also expressed in the epithelial cells of renal tubules, in the choroid plexus and in the retinal pigment layer, in erythroblasts and pinealocytes.
  • the HT7 protein can have considerable diagnostic and therapeutic importance. Accordingly, it was the object of the present invention to characterize this protein by purification, cloning and expression at the molecular level.
  • the invention thus relates to a new membrane protein with two immunoglobulin-like domains, the amino acid sequence shown in FIG. 3D, naturally occurring allelic variations of this amino acid sequence or a sequence homologous to this amino acid sequence with essentially the same biological properties and has functions.
  • the protein according to the invention has several glycosylation sites and, depending on its location, can be present in differently glycosylated forms.
  • the N-terminal amino acid sequences of the protein, purified from chicken brain capillaries, kidneys and erythroblasts, are identical.
  • Specific oligonucleotide primers could be constructed on the basis of partial information of the amino acid sequence, so that a cDNA clone was obtained by the polymerase chain reaction.
  • HT7 The nucleotide sequence of the HT7 protein reveals that it is a member of the iminunglobulin superfamily that contains two C2-like domains. In certain areas there is considerable homology to the v-fms oncogene. There was a region in the presumably cytoplasmic end of the protein found with clear homology to the p53 nuclear oncogene. So far, the sequence has not been discovered in membrane proteins. Furthermore, their biological meaning is still • unknown. On the basis of these results, it can be suggested that HT7 is a receptor which is involved in the cell-surface recognition at the blood-brain barrier. It can therefore be a valuable marker for these important processes at the blood-brain barrier. HT7 could also allow the specific transport of drugs and toxins into the brain.
  • HT7 protein For the characterization of suitable cells or tissues for the purification of the HT7 protein, monoclonal and polyclonal antibodies against immune-purified HT7 antigen were used (W. Risau, R. Hallmann, U. Albrecht and S. Henke-Fahle, EMBO J. 5, 3179-3183 (1986)). Such antibodies can be produced by the person skilled in the art using the method described above without major difficulties.
  • a protein with an apparent molecular weight of approximately 45 to 52 kD was found in extracts from chicken brain microvessels (45 kD) kidney (52 kD) and erythroblasts
  • the erythroblast cell line HD3 is a cell line transformed with the poultry erythroblastosis virus (H. Beug, G. Doederlein, C. Freudenstein and T. Graf, J. Cell. Physiol. 115, 295-309
  • the HT7 antigen was expressed on the cell surface of these cells in a manner similar to that of a chicken hen endothelial cell culture (Fig. 1B, C).
  • the erythroblast cell line HD3 is therefore a good source of the antigen.
  • the antigen was purified from erythroblast plasma membrane proteins, which were solubilized with Nonidet P40, with the aid of HT7 monoclonal antibodies Bromine-activated Sepharose 4B were bound.
  • the eluate from this affinity column was passed through a reverse phase C4-HPLC column.
  • the protein-containing peak fractions were analyzed in immunoblots for the presence of the HT7 antigen.
  • Figure 2A shows HPLC (a) and immunoblot (b) analysis of immunopurified HT7 antigen.
  • the only peak (4) which contained the antigen was analyzed by gel electrophoresis and silver training (FIG. 2B) and then sequenced using the gas phase amino acid microsequencing method. The N-terminal amino sequence was identified with
  • oligonucleotide primers were constructed and used for the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • a resulting amplified DNA fragment with the expected size of 120 bp was found, which hybridized with a pool of degenerate oligonucleotides, which was constructed using an internal sequence.
  • the other pool of degenerate oligonucleotides used as a control did not hybridize (Fig. 3B).
  • the amplified fragment was cloned into a vector and used to examine an erythroblast ⁇ gtll cDNA library. The investigation of 1.5 x 10 5 clones revealed 30 positive clones.
  • the cleavage of the signal peptide presumably occurs between the alanine (-1) - and threonine residue * -l), which would correspond to the N-terminal amino acid sequence obtained by amino acid sequence analysis.
  • a transmembrane region that contains a typical membrane anchor with basic amino acids extends from alanine (187) to isoleucine (207).
  • the hydropathy analysis of the predicted protein agrees with these interpretations (FIG. 3E).
  • the predicted molecular weight of the mature protein is 24075 D, which is in good agreement with the estimate of 25 kD that was obtained after gel electrophoresis of erythroblasts treated with tunicamycin (FIG. 2D).
  • the corresponding PTH amino acids were not identified at positions 21, 80 and 138, which indicates that at least these residues glycosylate in the mature protein could be.
  • 150 were identified by amino acid sequence analysis (underlined in FIG. 3D).
  • the predicted amino acid sequence of the cDNA clone matched all of the amino acid sequences obtained. Examination of the MIPSX database confirmed that this gene had not been previously sequenced.
  • the arrangement of the nucleotide sequence of the HT7 gene with published sequences allows the HT7 gene to be clearly classified into the immunoglobulin superfamily.
  • Two immunoglobulin-like domains with the characteristic ⁇ -sheet sandwich (predicted by Chou-Fassmann analysis), stabilized by disulfide bridges, are predicted from the analysis of homology regions and preserved cysteine residues (FIGS. 4A and D) .
  • a comparison of the immunoglobulin domains of the HT7 protein with other members of the immunoglobulin superfamily indicates that they are of the C2 type according to the criteria of Williams and Barclay, Ann. Rev. Immunol. 6, 381-405 (1988).
  • the total homology with a protein gp70 M.
  • leucine zipper which consists of 4 to 5 leucine residues repeated every 7th amino acid, is important for the functional dimerization of DNA-binding proteins (T. Kouzarides and E. Ziff, Nature 336, 646-651 (1988 ), WH Landshultz, PF Johnson and SL McKnight, Science 243, 1681-1688 (1988). It is known that the leucine zipper motif also occurs in membrane transport proteins and ion channels (K. McCormack et al., Nature 340, 103 (1989) and MK White and MJ Weber, Nature 340, 103-104 ( 1989)), but its meaning is questioned (V.
  • a new motif encoded in the predicted cytoplasmic end of the HT7 gene has homology to a sequence of the p53 nuclear oncogene (Fig. 4C) (T. Soussi et al., Nucl. Acids Res. 16, 11383 (1988)) . 5 consecutive amino acids are identical and some are conserved in the neighborhood (38% identity in 29 amino acids), so far this sequence has not been found in membrane proteins and their biological significance is unclear. Based on the homologies, a model for the structure of the HT7 protein is proposed (FIG. 4D).
  • the HT7 antigen is a new protein. Based on the available results, it is a receptor that is involved in cell surface recognition at the blood-brain barrier. The ligand is still unknown.
  • the HT7 antigen is a specific protein from chicken endothelial cells of the blood-brain barrier. It has not been found in other endothelial cells. It is therefore a marker for an organ-specific endothelium. However, it is not brain specific. Expression is also found in erythroblasts, epithelial cells of renal tubules, choroid plexus and retinal pigment layer. This suggests that the HT7 protein participates in transport processes into these cells.
  • HT7 protein can be a role in cell adhesion.
  • the antigen was concentrated in areas with cell-cell contact (Fig. IB).
  • ICAM cell adhesion molecules
  • N-CAM N-CAM
  • the HT7 protein is unlikely to be a growth factor receptor because (1) its expression does not match the growth status of the cell and (2) tyrosine phosphorylation sites are absent from the cytoplasmic end.
  • HT7 protein can be a valuable marker for elucidating these processes and their disruption under pathological conditions such as in brain tumors (DR Groothuise and NA Vick, TINS 5, 232-235 (1982)), Alzheimer's disease (CL Masters and K.
  • HT7 protein according to the invention could also be used for the specific transport of drugs in and possibly through the blood-brain barrier.
  • Microvessels were isolated from brain from adult chickens by a single pronase digestion (0.5% weight / volume in serum-free DMEM, one hour at 37 ° C.) from pieces of the cerebral cortex, those of meninges were cleaned. The microvessels settled after centrifugation (20 minutes, 1000 g) in 25% bovine serum albumin in saline, while neural tissue swam under these conditions. Microvessels, erythroblasts (H. Beug et al., J. Cell. Physiol.
  • Brain endothelial cells were cultured from embryo brain (day 18) microvessel fragments in DMEM, 10% calf serum and 1% bovine retina extract (containing fibroblast growth factors) as already described (W. Risau et al., J. Cell Biol. (1989)). Endothelial colonies were identified by the expression of FVIIIrAg-vWF and incubated with monoclonal HT7 antibodies (10 ⁇ g IgG per ml). They were then fixed using 4% buffered paraformaldehyde and stained with secondary rhodamine-labeled antibodies. Controls using monoclonal antibodies corresponding to isotype or preimmune serum in immunofluorescence and immunoblots were in each case negative.
  • FIG. 1A shows 70 ⁇ g protein from chicken brain microvessels (lane 1), kidney (2) and erythroblasts (3), which were separated by PAGE in a 7.5 to 17.5% polyacrylamide gel under reducing conditions and using polyclonal HT7 antibodies were immunoblotted.
  • Figures IB and C show indirect immunofluorescence from chicken brain endothelial cells (B) and erythroblasts (C).
  • Example 2
  • Erythroblasts were cultivated in bioreactors (1 1) with continuous stirring and harvested by centrifugation. From this, plasma membrane proteins were isolated (S. Hoffman et al., J. Biol. Chem. 257, 7720-7729 (1982)).
  • the HT7 antigen was purified from membrane proteins solubilized with Nonidet P40 by immunoaffinity chromatography, using HT7 monoclonal antibodies bound to Sepharose (2 mg IgG per ml packed gel, 50 ml gel). The bound material was eluted with buffered 3.5 M sodium thiocyanate, dialyzed, lyophilized, taken up in water and applied to a VYDAC-C4 reverse phase column (25 cm, 4.5 mm inner diameter).
  • the bound material was eluted using a linear gradient from 25 to 75% solvent B.
  • the peak fractions were lyophilized and analyzed in immunoblots (cf. Example 1).
  • the amino acid sequencing was carried out using an Applied Biosystems gas phase microsequencing apparatus. Solid phase peptide synthesis was performed in an Applied Biosystems Model 431A automated peptide synthesizer using Fmoc-protected amino acids.
  • the peptide was bound to keyhole limpet hemocyanin (H. Shapira et al., Proc. Natl. Acad. Sci. USA 81, 2461-2465 (1984)). Rabbits were immunized with this. The rabbits' sera were tested in immunoblots.
  • FIG. 2A shows a reverse phase C4-HPLC-Diagra ⁇ m_ ⁇ of the immunologically purified erythroblast HT7 antigen.
  • the proteins in the indicated peak fractions were lyophilized, electrophoretically separated on a 12% polyacrylamide gel and analyzed in immunoblots (FIG. 2B).
  • the antigen is contained in fraction 4, which elutes at 55% solution B (95% acetonitrile, 0.1% TFA).
  • FIG. 2B shows PAGE (lanes 1 and 2) and immunoblot analysis (lanes 3 and 4) of immune-purified and C4-HPLC-purified HT7 antigen from brain (1.3) and kidney (2.4) plasma membrane proteins.
  • FIG. 2D shows an immunoblot analysis of the HT7 antigen from brain microvessels using antibodies against the N-terminal peptide (lane 1). Munoblot analysis of the HT7 antigen after tunicamycin treatment (1 ⁇ g / ml for 24 hours) of erythroblasts is shown in lane 3.
  • FIG. 2E shows the amino acid sequences of the N-terminus, the CNBr fragment and of trypsin fragments of the HPLC-purified HT7 antigen.
  • X represents an unidentified amino acid.
  • Oligonucleotides were made with an Applied Biosystems DNA Synthesizer 381A.
  • CDNA complementary to mRNA from erythroblasts (C. Chen and H. Okayama, Mol. Cell. Biology 7, 2745-2752 (1987)) was produced according to known methods (U. Gubler, Nucl. Acids Res. 16, 2726 (1988)) and as a template for the PCR reaction (SI Girgis et al., Nucl. Acids Res. 16, 10371 (1988)), the degenerate sense and antisense primer molecules (see FIG. 3A) were used at the specified annealing temperatures (Fig. 3B).
  • the amplified DNA fragments were separated on 2.5% agarose gels, blotted on nylon membranes and hybridized with the 2 P-labeled test oligonucleotides.
  • the 120 bp fragment was eluted and cloned into the commercially available Bluescript KS vector.
  • An erythroblast ⁇ gtll cDNA library made in a manner known to those skilled in the art was tested using the 120 bp fragment. Inserts from two positive clones (out of a total of 30) were sequenced (F. Sanger et al., Proc. Natl. Acad. Sci. USA 72, 3918-3921 (1979)) as indicated in Figure 3C.
  • FIG. 3A shows the sequence of primers and test oligonucleotides for the PCR reaction based on the amino acid sequence of the CNBr fragment of HT7.
  • FIG. 3B shows pool B of the test oligonucleotides (SOb) which give a strong hybridization with the expected 120 bp fragment (arrow), whereas pool A (SOa) was negative.
  • FIG. 3C shows a restriction map of the cDNA clones pCHT7.1 and pCHT7.2. The arrows indicate the direction and scope of the nucleotide sequence analysis.
  • Figure 3D shows the nucleotide sequence of the HT7 gene. The two initiation codons and the suspected transmembrane region are boxed.
  • FIG. 3E shows a hydropathy blot of the suspected amino acid sequence of the HT7 protein below Using the algorithm of Kyte and Doolittle, J. Mol. Biol. 157, 105-132 (1982).
  • FIG. 4D shows the suspected leucine zipper motif. Leucine or isoleucine residues participating in the hypothetical zipper are framed.
  • FIG. 4C shows the homology of the HT7 protein with the oncoprotein p53.
  • FIG. 4D represents a possible structural model for the HT7 protein. The circles held together by disulfide bridges represent possible immunoglobulin domains. Assumed N-glycosylation sites are marked with small circles.
  • High molecular genomic chicken DNA was made from the liver of an adult chicken. Samples of 20 ⁇ g each were cleaved by restriction enzymes, applied on a 0.9% agarose gel and blotted on Genescreen nylon membranes (DuPont). 5 ⁇ g poly-A-RNA, isolated from the respective tissues (P. Chomczynski and N. Sacchi, Anal. Biochem. 162, 156-159 (1987)), was in a 0.9% formaldehyde denaturing agarose gel electrophoresed and blotted onto a Hybond-N nylon filter (Amersham).
  • the hybridization reactions were carried out in a 50% formamide solution, 5X SSC, 5X Denhardt's solution, 5% SDS and 100 ⁇ g / ml salmon sperm DNA at 42 ° C. for 12 hours. It was washed three times with 0.1 ⁇ SSC, 1% SDS for 15 minutes at 65 ° C.
  • the EcoRI / PstI fragment of the pCHT7.2 clone (FIG. 3A) was cloned into the vector pCMV (S. Andersson et al., J. Biol. Chem. 264, 8222-82229 (1989)) and thus COS- Cells transfected (P. Chomczynski and N. Sacchi, Anal. Biochem.
  • FIG. 5A shows a Southern blot analysis of chicken DNA digested with BamHI, EcoRI and PstI. It can be seen from this that the HT7 gene occurs only once in the chicken genome.
  • FIG. 5B shows a Northern blot analysis of poly-A RNA from the brain (lane 1), kidney (2) and erythroblasts (3). The blots were hybridized with the 120 bp fragment from FIG. 3C.
  • FIGS. 5C and D show the expression of the HT7 protein by transfected COS cells. Indirect immunofluorescence (C) and phase contrast (D) from COS cells using HT7 monoclonal antibodies were performed. 20 to 30% of the cells showed bright membranes and intracellular fluorescence. The magnification is 250 times.

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Abstract

L'invention concerne une protéine membranaire à deux domaines du type immunoglobuline, possédant (a) la séquence d'aminoacides représentée à la figure 3D, (b) une variante allélique, existant à l'état naturel, de cette séquence d'aminoacides, ou, (c) une séquence homologue à celle-ci, correspondant à une protéine ayant essentiellement les mêmes activités et fonctions biologiques, ainsi qu'un ADN de recombinaison codant pour la protéine selon l'invention, un procédé pour sa fabrication et son utilisation comme médicament.
PCT/EP1990/002000 1989-11-24 1990-11-22 Clonage et expression de la proteine ht7 specifique de la barriere hematoencephalique WO1991008232A1 (fr)

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DE19893938953 DE3938953A1 (de) 1989-11-24 1989-11-24 Klonierung und expression des fuer die blut-hirn-schranke spezifischen proteins ht7
DEP3938953.7 1989-11-24

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AU665599B2 (en) * 1991-11-08 1996-01-11 Hemosol Inc. Hemoglobins as drug delivery agents

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
The EMBO Journal, Band 5, Nr. 11, November 1986, IRL Press Ltd, W. Riseau et al.: "Brain induces the expression of an early cell surface marker for blood brain barrier-specific endothelium", Seiten 3179-3183 *
The EMBO Journal, Band 9, Nr. 7, Juli 1990, Oxford University Press, H. Seulberger et al.: "The inducible blood-brain barrier specific molecule HT7 is a novel immunoglobulin-like cell surface glycoprotein", Seiten 2151-2158 *

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