U.S. patent application number 09/898541 was filed with the patent office on 2002-06-13 for method and reagents for genetic immunization.
Invention is credited to Bartido, Shirley M., Houghton, Alan, Wang, Siqun, Xu, Yiquing.
Application Number | 20020072504 09/898541 |
Document ID | / |
Family ID | 21811039 |
Filed Date | 2002-06-13 |
United States Patent
Application |
20020072504 |
Kind Code |
A1 |
Houghton, Alan ; et
al. |
June 13, 2002 |
Method and reagents for genetic immunization
Abstract
DNA vaccines which incorporate genetic sequences encoding
sorting signals which direct an expressed antigen to a specific
cellular organelle facilitate loading of the antigen onto a Class I
or Class II MHC molecule for immune presentation. These vaccines
are a nucleic acid construct of a genetic sequence encoding a
protein or peptide antigen and a sorting signal which will direct
expressed antigen to the ER or endosomal-lysosomal compartments
within the cell. The resulting constructs can be used as naked DNA
vaccines, packaged in liposomes, or coated onto colloidal gold
particles. The construct might also be delivered in an expression
vector which is expressed in cells of the organism being
immunized.
Inventors: |
Houghton, Alan; (New York,
NY) ; Bartido, Shirley M.; (Jersey City, NY) ;
Xu, Yiquing; (New Rochelle, NY) ; Wang, Siqun;
(Wilmington, DE) |
Correspondence
Address: |
OPPEDAHL AND LARSON LLP
P O BOX 5068
DILLON
CO
80435-5068
US
|
Family ID: |
21811039 |
Appl. No.: |
09/898541 |
Filed: |
July 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09898541 |
Jul 2, 2001 |
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09230199 |
Jun 9, 1999 |
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09230199 |
Jun 9, 1999 |
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PCT/US97/12675 |
Jul 18, 1997 |
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60022710 |
Jul 26, 1996 |
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Current U.S.
Class: |
514/44R ;
514/1.2; 514/19.3; 514/3.7 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 2319/06 20130101; A61P 37/04 20180101; C07K 14/47 20130101;
C12N 9/0071 20130101; C12N 15/62 20130101; A61K 39/00 20130101;
A61P 31/00 20180101; C07K 2319/40 20130101; C12N 9/0059 20130101;
C07K 2319/04 20130101 |
Class at
Publication: |
514/44 ;
514/12 |
International
Class: |
A61K 048/00; A61K
038/17 |
Claims
1. A nucleic acid construct for genetic immunization comprising (a)
an antigen-coding region encoding an antigenic protein or peptide;
and (b) a sorting region encoding a protein or peptide which acts
as a sorting signal to direct intracellular transport of the
protein or peptide to the endosomes or the endoplasmic reticulum of
a cell.
2. The construct of claim 1, further comprising a linker region
disposed between the antigen-coding region and the sorting
region.
3. The construct according to claim 1 or 2, wherein the sorting
region is derived from the human brown locus protein, gp75; human
albino locus protein, tyrosinase; human silver locus protein, Pmel
17; or human pink eyed locus P-protein.
4. The construct according to claim 1 or 2, wherein the sorting
region encodes at least the peptide Glu Ala Asn Gln Pro Leu Leu Thr
Asp (SEQ ID NO. 1).
5. The construct according to claim 1 or 2, wherein the sorting
region encodes at least the peptide Glu Glu Lys Gln Pro Leu Leu Met
Asp (SEQ ID NO. 2).
6. The construct according to claim 1 or 2, wherein the sorting
region encodes at least the peptide Glu Asp Ser Pro Leu Leu (SEQ ID
NO. 3).
7. The construct according to claim 1 or 2, wherein the sorting
region encodes at least the peptide Glu Asp Thr Pro Leu Leu (SEQ ID
NO. 4).
8. The construct according to claim 1 or 2, wherein the sorting
region encodes at least the peptide sequence Pro Ser Arg Asp Arg
Ser Arg His Asp Lys Ile His (SEQ ID NO. 5).
9. The construct according to claim 1 or 2, wherein the sorting
region is a mutant form in which a glycosylation site present in a
corresponding wild type sorting region has been altered.
10. The construct according to any of claims 1 to 9, further
comprising a promoter region effective to permit expression of the
construct in mammalian cells.
11. The construct according to claim 10, wherein the promoter
region is selected from among the SV40 promoter, the CMV promoter
and the RSV promoter.
12. A vaccine for genetic immunization comprising a nucleic acid
construct according to any of claims 1 to 11.
13. The vaccine according to claim 12, wherein the nucleic acid
construct is packaged in a liposome.
14. The vaccine according to claim 12, wherein the nucleic acid
construct is coated on a colloidal gold particle.
15. The vaccine according to claim 12, wherein the nucleic acid
construct is incorporated into a viral expression vector.
16. A method for inducing an immune response to an antigen in a
mammal, comprising the step of administering to the mammal a
nucleic acid construct or vaccine according to any of claims
1-15.
17. A method for preparing a vaccine for genetic immunization
comprising the step preparing a nucleic acid construct according to
any of claims 1 to 11.
18. The method according to claim 17, further comprising the step
of packaging the nucleic acid construct in a liposome carrier.
19. The method according to claim 17, further comprising the step
of coating the nucleic acid construct on a colloidal gold
particle.
20. The method according to claim 17, wherein the nucleic acid
construct is incorporated into a viral expression vector.
Description
[0001] This application relates to improved reagents for use in
"genetic immunization," and to a method for genetic immunization
which makes use of these reagents to elicit a more potent immune
response.
[0002] The generation and regulation of immune response is a result
of a complex system of interactions between B- and T-lymphocytes,
circulating antibodies, and antigen presenting cells (APC). The
induction of humoral and cell-mediated immune responses to protein
antigens requires the recognition of the antigens by helper T (TH)
cells. The reasons for this is that helper T cell are necessary for
stimulating B-lymphocyte growth and differentiation, and for
activating the effector cells of cell-mediated immunity, including
macrophages and cytolytic T lymphocytes (CTLs). Briefly, foreign
antigen is processed by APCs which result in the generation of
antigen-derived peptide fragments bound to the major
histocompatability complex (MHC) Class I and Class II molecules
(referred to as human leukocyte antigens or HLA Class I and Class
II proteins in humans). These complexes which are found on the cell
surface of the APC are then presented to TH cells. Recognition of
the peptide-MHC complex by T cells is the initiating stimulus for T
cell activation. Thus, more efficient presentation of peptide-MHC
complex can lead to more efficient T cell activation. Activation
leads to the secretion of cytokines, proliferation, and regulatory
or cytolytic effector functions which all lead to immunity, in part
through the eradication of cells presenting antigen.
[0003] T cell-mediated eradication of cells expressing antigen can
be accomplished in three ways. First, humoral responses occur when
activated TH cells stimulate the proliferation and differentiation
of specific B cell clones to produce antibodies which eventually
eliminate cells expressing the antigen as well as extracellular
antigen. Second, cell-mediated responses occur when cytokines
activate T cells to differentiate into CTLs. The infected target
cell is then lysed by the CTL. Endogenous antigens, such as viruses
and tumor antigens, activate Class-I restricted CTLs, which lyse
cells producing these intracellular antigens. Third, nonspecific
responses occur when antigen-activated T cells secrete cytokines
that recruit and activate inflammatory cells such as macrophages
and natural killer cells that are not specific for the antigen.
Overall, therefore, T cells play a central role in recruiting a
broad immune response.
[0004] As used herein, the term "genetic immunization" refers to
the use of DNA as a vaccine to produce an immune response to the
protein or peptide antigen encoded by the DNA. Intramuscular
administration of naked DNA has been shown to elicit both humoral
and cellular immune response. The precise mechanism by which DNA
vaccines elicit an immune response is not known, although several
possibilities have been discussed. See Pardoll et al., "Exposing
the Immunology of Naked DNA Vaccines", Immunity 3: 165-169 (1995).
Regardless of the mechanism, however, the effectiveness of DNA
vaccines to produce both humoral and cellular immunity indicates
that naked DNA is expressed after administration, with the protein
or peptide product being presented as an antigen in association
with either Class I or Class II proteins.
[0005] The processing and presentation of antigens by Class I and
Class II molecules occurs in different organelles within the cells.
Specifically, the endoplasmic reticulum (ER) has been shown to be
the site for loading peptide antigens derived from the cytoplasm
onto Class I molecules, while the endosomes/lysosomes have been
shown to be the site for loading peptide antigens onto Class II
molecules. Thus, the type of immune response and the extent to
which an immune response is generated may depend in significant
measure on the amount of antigen reaching the ER and endosomal
loading sites. It would therefore be highly advantageous to be able
to direct and control the accumulation of antigen within a desired
location within the cell to provide optimum immune response.
[0006] It is an object of the present invention to control the
trafficking to and stability of selected antigens within specific
cellular organelles, and to use this method to provide for enhanced
genetic immunization.
[0007] It is a further object of the present invention to provide
DNA vaccines which incorporate genetic sequences encoding sorting
signals which direct the expressed antigen to a specific cellular
organelle and facilitate loading of the antigen onto a Class I or
Class II MHC molecule for immune presentation.
[0008] It is still a further object of the invention to provide a
method for genetic immunization utilizing DNA vaccines which
incorporate genetic sequences encoding sorting signals which direct
the expressed antigen to a specific cellular organelle and
facilitate loading of the antigen onto a Class I or Class II MHC
molecule for immune presentation.
SUMMARY OF THE INVENTION
[0009] These and other objects of the invention are achieved
through the construction of a genetic sequence encoding a protein
or peptide antigen and a sorting signal which will direct expressed
antigen to the ER or endosomal-lysosomal compartments within the
cell. The resulting constructs are useful as DNA vaccines, and can
be used as naked DNA, packaged in liposomes, or coated onto
colloidal gold particles. The construct might also be delivered in
an expression vector, for example a viral vector, which is
expressed in cells of the organism being immunized.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows three forms of an ovalbumin/gp75 fusion
protein;
[0011] FIG. 2 shows the induction of CD4+ T cell response using the
invention;
[0012] FIG. 3 shows the induction of an IgG response using the
invention;
[0013] FIG. 4 shows the affect of genetic immunization in
accordance with the invention on tumor growth in mice;
[0014] FIG. 5 shows a method for forming a nucleic acid construct
in accordance with the invention;
[0015] FIG. 6 shows the construction of three different plasmids
containing a construct in accordance with the invention; and
[0016] FIG. 7 shows a graphical representation of the immune
response generated upon immunization with a construct in accordance
with the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0017] In accordance with the present invention, nucleic acid
constructs for use in genetic immunization procedures are prepared
which comprise
[0018] (a) an antigen-coding region encoding an antigenic protein
or peptide; and
[0019] (b) a sorting region encoding a protein or peptide which
acts as a sorting signal to direct intracellular transport of the
protein or peptide to the endosomal-lysosomal compartments or their
transport to/retention in the endoplasmic reticulum of a cell. As
used herein, the term "nucleic acid construct" reflects the fact
that the material is produced from component parts that are spliced
together from different sources and excludes, for example, a DNA
molecule encoding a naturally occurring protein that includes both
an antigenic determinant and a sorting signal region.
[0020] The antigen-coding region of the nucleic acid polymers of
the invention is selected to encode for one or more desired
antigenic determinants of a protein or peptide of interest. Thus,
the antigen-coding region may encode an entire protein or peptide,
or an immunogenic portion thereof associated with a selected
epitope of the protein or peptide.
[0021] The sorting region employed in the nucleic acids polymers of
the invention is selected to provide a peptide region that directs
intracellular transport of an expressed protein or peptide to a
desired intracellular location. Suitable sorting signals for
directing intracellular transport of the expressed antigen to the
endosomes include the following molecules: the signal from human
gp75 (brown locus protein) which includes the signal region
[0022] Glu Ala Asn Gln Pro Leu Leu Thr Asp; SEQ ID No. 1
[0023] the signal from human tyrosinase (albino locus protein)
which includes the signal region
[0024] Glu Glu Lys Gln Pro Leu Leu Met Asp; SEQ ID No. 2
[0025] the signal from human gp100 (silver locus protein, Pmel 17)
which includes the signal region
[0026] Glu Asp Ser Pro Leu Leu; and SEQ ID No. 3
[0027] the signal from human P-protein (pink eyed locus) which
includes the signal region
[0028] Glu Asp Thr Pro Leu Leu SEQ ID No. 4
[0029] as described in Vijayasaradhi et al., "Intracellular Sorting
and Targeting of Melanosomal Membrane Proteins: Identification of
Signals for Sorting of the Human Brown Locus Protein, GP75" J. Cell
Biology 130: 807-820 (1995).
[0030] Suitable sorting signals for directing intracellular
transport of the expressed antigen to the endoplasmic reticulum (or
retention therein) include the signal region
[0031] Pro Ser Arg Asp Arg Ser Arg His Asp Lys Ile His SEQ ID No.
5
[0032] which has been shown to retain a viral glycoprotein in the
endoplasmic reticulum. Rose et al. "Altered cytoplasmic domains
affect intracellular transport of the vesicular stomatitis virus
glycoprotein" Cell 34: 513 (1993); Bartido et al. "Processing of a
viral glycoprotein in the endoplasmic reticulum for class II
presentation" Euro. J Immunol. 25: 22111-2219 (1995).
[0033] Mutant forms of naturally-occurring sorting
signal-containing proteins and peptides may also be used as the
sorting region of the invention. Such mutants can alter protein or
peptide trafficking by making the protein or peptide more unstable
in a particular compartment in the cell, including the ER and
endosome/lysosome. For example, because glycosylation plays an
important role in stabilizing endocytic membrane proteins within
different cellular compartments, glycosylation mutants can be used
to more closely control the intracellular transport of the
expressed antigen/sorting signal product to induce the desired form
of immune response. On any given protein, there will generally be
multiple glycosylation sites, with each site being of different
importance in its effect on the transport and degradation of the
protein. For example, in the case of mouse gp75, there are five
N-glycosylation sites, one of which strongly effects the resistance
to protease digestion and two others of which are important for
permitting export of the protein from the endoplasmic reticulum.
Other mutants forms, for example mutants forms which disrupt the
sorting signal regions described above, particularly the Pro Leu
Leu motif, can also be used in the method of the invention.
Constructs which have a sequence which is the same as the wild-type
sequence for a sorting signal, or which are a mutant variant of
such a wild-type, or which are synthetically generated to encode
the same protein/peptide sequence as the wild-type or mutant
variants based upon degeneracy of the nucleic acid code are
referred to in the specification and claims hereof as being
"derived from" the wild type protein or peptide.
[0034] Identification of suitable mutants can be identified by
creation of the desired mutant, for example by site-directed
mutagenesis, followed by testing of each mutant in a model system.
For example, in the case of gp75, the two mutant forms shown in
FIG. 1 were compared to the wild-type mutant. The sorting signal
peptide labeled as "Deletion" has a deletion mutation introduced in
the region spanning Asn 511 to Asp 517, which results in the
deletion of the sorting signal region. The sorting signal peptide
labeled L2A differs from the wild-type by a single base
substitution, Leu514 to Ala514 which disrupts the Pro Leu Leu motif
of the sorting signal.
[0035] Testing of the ova/gp75 constructs shown in FIG. 1 for their
ability to induce an immune response to ova in mice showed
differential results depending on the sorting signal employed. As
shown in FIGS. 2 and 3, the construct containing the wild-type
sequence produced a MUCH greater CD4.sup.+ T Cell response, while
the constructs containing the mutant sequence produced higher
levels of IgG response. Both the fusion protein containing the
wild-type sequence and the fusion protein containing the L2A
mutation were effective to provide protection against tumor cells
expressing ovalbumin. (FIG. 4).
[0036] Glycosylation-mutants of sorting signals can be similarly
prepared and tested for their ability to direct the trafficking of
the expressed fusion proteins to desired regions within the cell.
Mutations at two of the five identified glycosylation sites on
mouse gp75 (Asn304 and Asn385) produced proteins which are
apparently retained and degraded in the endoplasmic reticulum. Such
mutant sorting signals can be used in fusion proteins in accordance
with the invention to selectively generate peptides for the MHC
Class I pathway. A third mutant form (Asn350) was transported from
the ER to the Golgi apparatus at a similar rate to the wild-type
but exhibited a markedly decreased half-life, being very unstable
in endosomes. Such mutants can be used in fusion proteins to direct
an MHC Class II response, and the enhanced degradation of the
sorting signal may generate more peptides for presentation through
this pathway. Mutation at Asn181 of mouse gp75 impacted the rate of
transport with the result that the mutant protein tended to
localize in the endosomal/lysosomal structures of the transfectants
and not in the Golgi apparatus. Fusion proteins with sorting
signals of this type can also be used to direct an MHC Class II
response to the antigen.
[0037] The antigen-coding region and the sorting region are
combined into a single nucleic acid polymer which may optionally
contain a linker region to ensure proper folding of the encoded
fusion protein. One suitable technique for this process utilizes
initial separate PCR amplification reactions to produce the two
regions, each with a linker segment attached to one end, followed
by fusion of the two amplified products in a further PCR step using
the general scheme shown in FIG. 5. This technique is referred to
as linker tailing. Of course, it will be appreciated that other
techniques and variations on this technique can be used. For
example, when either the antigen-coding region or the sorting
region is fairly short, the region may be chemically synthesized
and coupled to the other region by ligation. Suitable restriction
sites may also be engineered into regions of interest, after which
restriction digestion and ligation is used to produce the desired
fusion-protein encoding sequence.
[0038] After synthesis, the nucleic acid polymer containing both
the antigen-coding region and the sorting region is combined with a
promoter which is effective for expression of the nucleic acid
polymer in mammalian cells. This can be accomplished by digesting
the nucleic acid polymer with a restriction endonuclease and
cloning into a plasmid containing a promoter such as the SV40
promoter, the cytomegalovirus (CMV) promoter or the Rous sarcoma
virus (RSV) promoter. The resulting construct is then used as a
vaccine for genetic immunization. The nucleic acid polymer could
also be cloned into plasmid and viral vectors that are known to
transduce mammalian cells. These vectors include retroviral
vectors, adenovirus vectors, vaccinia virus vectors, pox virus
vectors and adenovirus-associated vectors.
[0039] The nucleic acid constructs containing the promoter,
antigen-coding region and sorting region can be administered
directly or they can be packaged in liposomes or coated onto
colloidal gold particles prior to administration. Techniques for
packaging DNA vaccines into liposomes are known in the art, for
example from Murray, ed. "Gene Transfer and Expression Protocols"
Humana Pres, Clifton, N.J. (1991). Similarly, techniques for
coating naked DNA onto gold particles are taught in Yang, "Gene
transfer into mammalian somatic cells in vivo", Crit. Rev. Biotech.
12: 335-356 (1992), and techniques for expression of proteins using
viral vectors are found in Adolph, K. ed. "Viral Genome Methods"
CRC Press, Florida (1996).
[0040] The compositions of the invention are preferably
administered intradermally, subcutaneously or intramuscularly by
injection or by gas driven particle bombardment, and are delivered
in an amount effective to produce an immune response in the host
organism. The compositions may also be administered ex vivo to
blood or bone marrow-derived cells (which include APCs) using
liposomal transfection, particle bombardment or viral infection
(including co-cultivation techniques). The treated cells are then
reintroduced back into the mammal to be immunized. While it will be
understood that the amount of material needed will depend on the
immunogenicity of each individual construct and cannot be predicted
a priori, the process of determining the appropriate dosage for any
given construct is straightforward. Specifically, a series of
dosages of increasing size, starting at about 0.1 ug is
administered and the resulting immune response is observed, for
example by measuring antibody titer using an ELISA assay, detecting
CTL response using a chromium release assay or detecting TH
response using a cytokine release assay.
[0041] The invention will now be further described and illustrated
by was of the following, non-limiting examples.
EXAMPLE 1
[0042] To demonstrate the creation of a nucleic acid construct in
accordance with the invention, a construct having an antigen-coding
region encoding chicken ovalbumin and a sorting region derived from
murine gp75 was produced. In the construct, the chimeric protein of
full length ovalbumin and the C-terminal region of gp75 containing
the sorting sequence
[0043] Glu Ala Asn Pro Leu Leu Thr Asp SEQ ID No. 1
[0044] are connected with a nine amino acid linker,
[0045] Ser Gly Gly Ser Gly Gly Ser Gly Gly. SEQ ID No. 6
[0046] The construct was prepared using a series of PCR reactions.
First, the ovalbumin gene coding amino acids 1-386 was amplified
from pAc-neo-OVA (Moore et al., "Introduction of Soluble Protein
into the Class I Pathway of Antigen Processing and Presentation"
Cell 54: 777-785 (1988)) with the primer pair
[0047] 5'-CGCCACCAGACATAATAGC-3' and SEQ ID No. 7
[0048] 5'-GCCTCCTGAACCTCCGGAACCACCAGAAGGGGAAACACATCTGCC-3'. SEQ ID
NO. 8
[0049] The transmembrane and cytoplasmic domains of gp75, amino
acid 488-539, were then amplified out from pSVK3-mpg75
(Vijayasaradhi et al., J. Cell Biol. 130: 807-820 (1995) using
primers
[0050] 5'-TCTGGTGGTTCCGGAGGTTCAGGAGGCATCATTACCATTGCTGTAGTG-3' SEQ
ID No. 9
[0051] and 5'-GGTTGCTTCGGTACCTGCTGCG-3'. SEQ ID No. 10
[0052] The PCR products from these two amplification were purified
and subjected to a second round of PCR using primers
5'-CGCCACCAGACATAATAGC-3- ' (SEQ ID NO. 11) and
5'-GGTTGCTTCGGTACCTGCTGCG-3' (SEQ ID No. 12). (See FIG. 5) The
second phase of the PCR fused the ova and gp75 sorting region with
the designed linker in between. Thus, the construct has a combined
open reading frame of 1365 base pairs capable of coding a protein a
protein of 455 amino acids which includes 386 amino acids from
ovalbumin, 9 amino acids from the linker and 60 amino acids from
gp75.
[0053] The construct was digested with EcoR1 and Kpn1 and cloned
into pSVK3 (Pharmacia/LKB Ltd.), pBK-CMV and pBK-RSV (Stratagene
Inc.) separately as shown in FIG. 6. These constructs have been
sequenced and their structures have been confirmed.
[0054] To construct targeting derivative mutants, PCR primers
containing mutations were synthesized. They are
5'-CTCAGCATAGCGTTGATAGTGATTCTTGGTGCT- TCTAGAACG-3' SEQ ID No 13 and
5'-CGTTCTAGAAGCACCAAGAATCACTATCAACGCTATGCTGA- G-3' (SEQ ID No. 14)
for the deletion of Asn511 to Asp517 mutant. The primer pair
5'-GAGTGCAGGCTGGTTGGCTTC-3' (SEQ ID No. 15) and
5'-CCTGCACTCACTGATCACTAT3' (SEQ ID NO. 16) are used to construct
the Leu514 to Ala514 mutant (FIG. 1). These constructs have been
sequenced and their structures have been confirmed.
EXAMPLE 2
[0055] Expression of the fusion protein as well as that of
ovalbumin alone was examined by utilizing plasmids that contained
the encoding DNA under different promoters. The DNA was transiently
transfected into mouse L cells or monkey COS cells by calcium
phosphate precipitation or DEAE-chloroquine methods. The cells
(1.times.10.sup.5) were plated on a 8-well chamber slide (Nunc,
Inc.) and incubated for 24 hours. Cells were then transfected with
0.5-1.0 .mu.g DNA by known standard calcium phosphate or DEAE
methods. After transfection, cells were allowed to grow for 24-48
hours prior to determining the intracellular localization of
ovalbumin in the transfected cell.
[0056] Detection of the protein was carried out by
immunofluorescent staining of the antigen. Cells were washed with
cold phosphate buffered saline (PBS), fixed with 2% formaldehyde,
permeabilized with methanol at -20.degree. C. and then incubated
with the monoclonal antibody (mAb) OVA-14 (BioMaker Inc.). The
cellular localization of the antigen was then visualized by
staining with a secondary antibody, FITC-conjugated goat anti-mouse
antibody (Dako, Inc.). The cells were observed using a Nikon
microscope and photographed using back and white film, ISO100.
[0057] Expression of the protein ovalbumin or of the fusion protein
gp75-ova was observed upon transfection of the DNA constructs into
cells using all three promoters (SV40, CMV and RSV). Furthermore,
when plasmids which included gp75 sorting region were introduced
into the cells, localized vesicular immunofluorescent staining was
observed consistent with endosome-lysosome localization. In
contrast, when the control plasmids without the gp75 sorting region
were introduced, a more diffuse cytoplasmic staining pattern
typical of staining of Golgi/ER localization was observed. Thus,
incorporation of the gp75 sorting region dramatically changed the
intracellular trafficking of a protein, ovalbumin, destined for the
secretory pathway to a protein contained in a vesicular compartment
in the endocytic pathway.
EXAMPLE 3
[0058] To test the ability of the constructs encoding the OVA/gp75
fusion protein to act as a vaccine for genetic immunization,
(C57BL/6xBalb/c) F1 mice were immunized with respective DNA
plasmids purified by using QIAGEN ion-exchange columns (Qiagen,
Inc.). DNA (100 .mu.g in 100 .mu.l of a 25% sucrose solution in
PBS) was injected subcutaneously at day 0 and day 14. Blood samples
were collected at day 14 and day 28.
[0059] Antibody response was monitored using an ELISA assay.
Chicken ovalbumin (Sigma, Inc.) was used as the antigen and plated
in a 96-well plate overnight at 4.degree. C. The diluted serum
samples were then added to the plate and incubated for 1 hour at
room temperature. After washing, the secondary antibody, alkaline
phosphatase-conjugated goat anti-mouse IgG, was added and plate was
incubated for 1 hour. Color development was achieved upon addition
of the Sigma Fast p-nitrophenyl substrate. Reaction was terminated
with the addition of 3N NaOH. Absorbance in the different wells was
obtained using the BioRad EIA Reader 2550. Results are shown in
FIG.7. As can be seen, the genetic immunization technique using the
constructs of the invention was effective to produce antibodies to
ovalbumin. Corresponding plasmid constructs with different
promoters also elicited an immune response, although not as
strongly as that seen when using the SV40 promoter.
EXAMPLE 4
[0060] CBF1 mice were immunized with DNA plasmids purified by the
QIAGEN ion-exchange columns (Qiagen, Inc.). To prepare bullets for
immunization, 50 mg of 0.95-2.6 .mu.m gold particles (Auragen,
Inc.) were mixed with 0.05-0.1 M spermidine, 100 .mu.g of plasmid
DNA was added to the mixture, and 1.0-2.5 M CaCl.sub.2 was added
dropwise while vortexing. After precipitation, the gold/plasmid DNA
complex was washed three times with cold 100% ethanol. Seven ml of
ethanol was added to the pellet to achieve a bead loading rate of
0.5 mg gold and 1.0 .mu.g plasmid DNA per injection. The
gold/plasmid DNA solution was then instilled into plastic
Tefzel.RTM. tubing, the ethanol gently drawn off, and the tube
purged with nitrogen gas at 400 ml/min for drying. The tube was cut
into 0.5 inch bullets and these were used for immunization. For
cutaneous immunizations, all mice were anesthetized with Metofane
inhalation (Pitman-Moore, Mundelein, Ill.). Abdominal hair was
removed with Nair.RTM. depilatory cream (Carter-Wallace, New York,
N.Y.), so that depilated abdominal skin was exposed for
immunization. The bullets were placed into a hand-held
helium-driven gene gun (Auragen, Inc.). Animals were immunized by
delivering the gold beads in one bullet into each abdominal
quadrant, for a total of four injections per immunization. Each
injection delivered 1 .mu.g DNA and therefore a total of 4 .mu.g
DNA per mouse each immunization. Each bullet was delivered to the
abdominal skin at a helium pressure of 400 pounds per square
inch.
[0061] In vivo antibody response. Indirect ELISA assays were
performed to monitor the antibody response. CB6F1 mice were
immunized with different plasmid constructs by gene gun once a week
for four weeks and a boost at week 6. Serum samples were collected
at weekly intervals. Purified chicken ovalbumin (Sigma, Inc.) was
used as the antigen and plated 50 .mu.g each well in a 96-well
plate overnight at 4.degree. C. The diluted serum samples were then
added to the plate and incubated for 1 hour at room temperature.
After washing, the second antibody goat anti-mouse IgG conjugated
with alkaline phosphatase (Sigma, Inc.) was added and incubated for
1 hour at 37.degree. C. The plates were developed using the Fast
p-Nitrophenyl phosphate substrate (Sigma, Inc.) and the reactions
were terminated with the addition of 3N NaOH. The absorbance at 605
nm were obtained by the BioRad EIA Reader 2550 (BioRad Inc.). The
positive control group immunized with naked DNA containing the full
length ovalbumin generated strong response within 2 weeks (FIG. 3).
Mutants with a disrupted (L2A) or deleted (del) sorting signal also
generated antibody response although the response appeared to be
delayed comparing to the wild type ovalbumin. Interestingly, the
ova/gp75 fusion protein failed to generate an antibody response
under the particular immunization protocol. The reason for that is
not known. But it is conceivable that most of the fusion protein is
sequestered in the cell due to its retention signal at the
c-terminus and not efficiently recognized by the B cells.
[0062] CD4+ T cell proliferative assay. A proliferation assay was
carried out to monitor the efficiency of in vivo priming of CD4+ T
cells by the different DNA constructs. CB6F1 mice were immunized
once a week for two weeks by gene gun and at day 14 the mice were
sacrificed. CD4+ T cells were purified from pooled splenocytes
using a CELLECT.TM..PLUS column (Biotex Laboratories, Inc.). The
purified CD4+ T cells (3.times.10.sup.5) were in vitro stimulated
by incubation with syngeneic naive splenocytes (1.times.10.sup.5)
pulsed with the denatured ovalbumin at different concentrations for
4 days at 37.degree. C. On day 4, 100 .mu.Ci of .sup.3H-TdR was
added to each well and the cpm is counted after 16-18 hours. The
proliferation response is expressed as the net cpm subtracting the
background (FIG. 2). The ova/gp75 fusion efficiently primed T cells
in vivo suggesting the endogenous processing and presentation of
the fusion protein. Moreover, melanosomal targeting mutants, L2A
and Del, did not prime well demonstrating the requirement of the
targeting signal for the function of the fusion protein in
stimulating CD4+ T cell prliferation.
[0063] Tumor protection. CB6F1 mice were immunized weekly for two
weeks with pBK-CMV vector alone, the vector contaning the ova-gp75
construct of FIG. 1 or the vector contaning the ova/L2A construct.
On day 14, the immunized mice were challenged by injecting
subcutaneously with 1.times.10.sup.6 MO4 melanoma cells, a B16
melanoma cell line transfected with the full length ovalbumin. Mice
were checked for tumor growth every other day over a period of 3
weeks. The "no treatment" group (n=10) all developed palpable tumor
within 2 weeks. Similarly, of the mice immunized with the vector
alone (n=10), all but one developed tumor. None of the mice
immunized with the vecotr encoding the ova/gp75 fusion protein
(n=10) developed tumor and only one out of ten developed tumor in
the L2A group (FIG. 4). This result clearly shows that the immune
response elicited by immunization with fusion protein construct can
lead to protection of tumor challenges in vivo.
[0064] To test the whether immunization by this method induced
immunologic memory, mice immunized with the fusion DNA were
re-challenged with tumor MO4 or B16 melanoma (the parent melanoma
cell line of MO4 that does not express the antigen ovalbumin) five
weeks after the last immunization. None of the five mice challenged
with MO4 developed tumor when observed for at least six weeks, and
two of the five mice challenged with B16 parental melanoma did not
develop tumor over at least 6 weeks. All five unimmunized mice
challenged with B16 developed tumors within 10-14 days. The sorting
signal was required for protection against B16 parental tumor. Five
mice immunized with the DNA construct containing a mutant sorting
signal (L2A) all developed tumor when challenged with B16. The
sorting signal was also required for potent immunological memory,
because one out of four mice immunized with construct containing
mutant sorting signal (L2A) developed tumor with MO4 tumor
challenge. This experiment show that immunization with DNA
constructs containing the tyrosinase family sorting signal can
provide long lasting memory against the antigen and can even
provide a broader protection against tumor challenge in tumors that
do not express the antigen.
EXAMPLE 5
[0065] To identify the sorting signal, constructs were made having
an antigen-coding region encoding the extracellular domain of the T
lymphocyte surface glycoprotein CD8, and a sorting region
containing the cytoplasmic tail of the human gp75 (amino acids 497
to 537) or the cytoplasmic tail and the transmembrane domains (TM)
of human gp75 (amino acids 477 to 537). To make these constructs, a
full-length 2.8 kb EcoRI fragment was isolated from a human
melanoma cDNA library and subcloned into the unique EcoRI site of
eukaryotic expression vectors pCEXV3 (Bouchard et al., J. Exp. Med.
169: 2029-2042 (1989)) or pSVK3.1 (a derivative of vector pSVK3
obtained by deletion of the Sac I fragment within the multiple
cloning site), or SmaI site of pSVK3 (Pharmacia LKB, Piscataway,
N.J.) following a fill-in reaction with Klenow fragment of DNA
polymerase (New England Biolabs, Beverly, Mass.). The orientation
of the cloned insert was determined by restriction analysis and
confirmed by dideoxy chain termination sequencing method (Sequenase
Kit, US Biochemicals, Cleveland, Ohio) using an oligonucleotide
primer complementary to the vector sequences upstream of the
cloning site.
[0066] Mouse L cell fibroblasts were transfected with plasmid
containing gp75 cDNA and pSV2neo. Transfected clones were isolated
by selecting for growth in the antibiotic G418 (1 mg/ml; Gibco BRL,
Gaithersburg, Md.), and screened for gp75 expression by
immuno-fluorescence staining with the mAb TA99 (Vijayasaradhi et
al., Exp. Cell Res. 171: 1375-1380 (1991)).
[0067] The plasmid EBO-pCD-Leu2 containing human CD8.alpha. cDNA
was obtained from American Type Culture Collection (Margolskee et
al., 1988). The 2.3 kb BamHI fragment from this plasmid was
isolated, made blunt-ended with Klenow fragment and cloned into the
SmaI site of the expression vector pSVK3. The orientation of the
cDNA insert in the recombinant plasmids in E. coli DH5.alpha. was
analyzed by appropriate restriction enzyme digestions, and
confirmed by DNA sequencing.
[0068] Chimeric cDNAs encoding fusion proteins CD8/gp75(TM+Cyt) and
CD8/gp75(Cyt) were constructed by the following methods. First,
appropriate restriction sites at or near the TM/Cyt junction of
CD8, and lumenal/TM and TM/Cyt junctions of gp75 were generated by
site-directed mutagenesis (Kunkel et al., 1987) using Mutagene kit
(BioRad Laboratories, Hercules, Calif.). Specifically, a mutant
gp75 plasmid pSVgp75RV was generated by introducing an EcoRV
restriction site at nucleotide 1560 (lumenal/TM junction) of gp75
cDNA in plasmid pSVK3 using the mutagenic oligonucleotide
[0069] 5'-TACTGCTATGGCAATGA TATCAGGTACACTA-3 SEQ ID No. 17
[0070] (mutations introduced are shown in bold and underlined).
This resulted in the conversion of glutamic acid at position 477
(amino acids numbered starting with the methionine coded by the
initiation codon) to aspartic acid. Mutant plasmids pSVgp75H and
pSVleu2H were generated by introducing a HindIII restriction site
in gp75 cDNA at nucleotide 1627, (gp75 TM/Cyt junction) and at
nucleotide 706 (CD8 TM/Cyt junction) in CD8 cDNA using the
mutagenic oligonucleotides
[0071] 5'-GCGTCTGGCACGAAGCTTATAAGAAGCAGT-3' and SEQ ID No. 18
[0072] 5'-GTCTTCGGTTCCTAAGCTTGCAGTAAAGGGT-3', SEQ ID No. 19
[0073] respectively. This resulted in conversion of leucine at
position 500 to lysine and isoleucine at 501 to leucine in gp75;
and asparagine at 207 to lysine and histidine at 208 to proline in
CD8. Mutants were first identified by appropriate restriction
enzyme digestion and confirmed by sequencing the relevant regions
of the plasmids using a Sequenase sequencing kit. Transient
expression in mouse fibroblasts and immunofluorescence analysis
with mAbs TA99 (anti-gp75) and OKT-8 (anti-human CD8) showed that
intracellular staining of mutant proteins was identical to the
distribution of wild type counterparts, I. e., punctate cytoplasmic
staining of gp75 and cell surface expression of CD8.
[0074] Plasmid pSVgp75RV was digested with EcoRV and XbaI to
produce a .apprxeq.1.2 kb fragment containing the TM+Cyt sequence
and 3' untranslated sequence of gp75 cDNA including part of the
multiple cloning site sequences of the vector; plasmid pSVleu2H was
digested with EcoRV and XbaI and the large .apprxeq.4 kb plasmid
DNA fragment lacking TM and Cyt sequences of CD8 cDNA was isolated.
The 1.2 kb EcoRV-XbaI gp75 fragment was ligated with the large
EcoRV-XbaI pSVleu2H fragment to generate a plasmid construct
encoding the fusion protein CD8/gp75 (TM+Cyt). Similarly, a
.apprxeq.1 kb HindIII-XbaI gp75 cDNA fragment (containing gp75 Cyt
and 3' untranslated sequences), and a .apprxeq.4 kb HindIII-XbaI
CD8 cDNA plasmid fragment (lacking the cytoplasmic tail sequences
of CD8) were isolated, respectively, from plasmids pSVgp75H and
pSVleu2H, and ligated to generate the fusion protein CD8/gp75(Cyt).
Regions of the plasmids at the CD8/gp75 junctions were sequenced
from at least two independent clones to confirm the restoration of
the reading frame. Large scale plasmid preparations (Quiagen, Inc.,
Chatsworth, Calif.) were further verified by restriction enzyme
digestions for the presence of enzyme sites unique to gp75 and CD8
at appropriate regions in the chimeric plasmids.
[0075] pSVK3.1gp75, was utilized to generate carboxyl terminal
deletion mutants. The restriction enzyme site BglII at nucleotide
2000 of gp75 cDNA is a unique site within the plasmid pSVK3.1.
Plasmid pSVK3.1gp75 (10 .mu.g) was linearized by digestion with 40
units of BglII in a 50 .mu.l reaction for 3 h at 37.degree. C.
Linearized .apprxeq.6.7 kb DNA was then digested for 3-4 min with
Bal 31 nuclease (1 unit enzyme/.mu.g DNA) in 50 .mu.l reaction.
Digested DNA was immediately extracted with phenol:chloroform to
inactivate and remove the nuclease, and the ends were filled in by
Klenow fragment of DNA polymerase I to increase the population of
blunt-ended molecules (Sambrook et al., 1989). Klenow fragment was
inactivated by heating at 75.degree. C. for 10 min, and a
suppressible reading frame termination linker containing
restriction site NheI, 5'-CTAGCTAGCTAG-3' (Pharmacia), was ligated
to the blunt-ended, truncated pSVK3.1gp75 DNA molecules with 1 unit
of T4 DNA ligase in 20 .mu.l reaction for 3 h at room temperature.
The ligation mixture was used to transform E. coli strain
DH5.alpha.. Ampicillin-resistant bacterial colonies were analyzed
by agarose gel slot lysis method for the presence of plasmid DNA of
appropriate size. Plasmid DNA from 15 transformants was isolated,
analyzed by restriction enzyme digestion, and partially sequenced
to determine the number of bases deleted from the carboxyl terminus
and to confirm the addition of termination linker.
[0076] A transient transfection method was developed and optimized
to study the intracellular distribution of gp75 expressed by mutant
constructs. Briefly, 2-4.times.10.sup.4 SK-MEL-23 clone 22a
melanoma cells and mouse L cells fibroblasts were plated in 8-well
LabTek chamber slides. The cells were transfected with plasmid DNA
by calcium phosphate precipitate method for 16-24 h, and then
allowed to accumulate the expressed protein for 12 to 48 h which
was evaluated by immunofluorescence microscopy and
immunoelectronmicroscopy.
[0077] For immunofluorescence microscopy, cells on the 8-well glass
slides were fixed with formaldehyde, followed by methanol, and
stained with gp75 specific mouse mAb TA99 or OKT-8 followed by
FITC-conjugated anti-mouse IgG. Cells were examined under Nikon
Optiphot fluorescence microscope and photographed using Kodak
Ektachrome film.
[0078] For immunoelectronmicroscopy, mAb TA99 directly conjugated
to 10 nm gold particles was used for localization of gp75 by
immunoelectron microscopy. Colloidal gold was prepared as described
(Smit and Todd, 1986) and mAb TA99-gold conjugate was prepared
according to Alexander et al., 1985. Human melanoma SK-MEL-19 cells
were fixed with 0.2% glutaraldehyde in 0.1 M phosphate buffer, pH
7.4, infused with 2.3 M sucrose in PBS and the cell pellet was then
frozen in liquid nitrogen. Ultrathin sections were cut and
collected on formvar-carbon coated nickel grids. The sections on
the grids were incubated in 0.5% BSA in PBS to block nonspecific
protein binding sites and then stained with mAb TA99 conjugated to
10 nm gold particles. Washing and staining of the sections was
performed according to Griffiths et al., 1983. Sections were
observed on a Jeol 100CX electron microscope.
[0079] These experiments showed that the expressed proteins from
constructs having a sorting region that included the 36 amino acid
cytoplasmic tail of human gp75 (with or without the transmembrane
region) were localized to the juxtanuclear region of the cells, and
there was little or no staining of other cytoplasmic structures of
the plasma membrane. This pattern showed localization of the
expressed protein in the Golgi region and possible other organelles
such as late endosomes and lysosomes present in the Golgi region.
It was further determined, however, that the absence of cell
surface staining which would be expected because of the presence of
the CD8 portion of the chimeric protein is probably the result of
protease degradation of the CD8 within the protease-rich endosomes
and lysosomes.
EXAMPLE 6
[0080] The role of specific N-glycans in determining stability of
an endocytic membrane protein within different cellular
compartments was investigated. The tyrosinase family of
glycoproteins has multiple conserved potential N-linked
glycosylation sites. The mouse brown locus protein, gp75, is a
prototype of the TRP family. We examined how N-linked glycosylation
on gp75 plays a role in maintaining the stability of this protein
as it is transported through different compartments, by
systemically eliminating each N-linked glycosylation sites.
[0081] An 1.8 Kb EcoR I fragment containing the full length mouse
gp75 cDNA was isolated from pMT4 plasmid (kindly provided by Dr. T.
Shibahara, Tohoku University School of Medicine, Japan), and
subcloned into the unique EcoR I site of eukaryotic expression
vector pSVK3.1 to generate pSVK3.1-mgp75. pSVK3.1 is a derivative
of pSVK3 (Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.),
modified by removing the Sac I fragment within the multiple cloning
sites. The orientation of the insert was determined by restriction
enzyme analysis and confirmed by DNA sequencing using Sequenase Kit
(US Biochemicals, Cleveland, Ohio). The Muta-gene Phagemid in vitro
mutagenesis kit (Bio-Rad, Melvile, N.Y.) was used to create Asn to
Gln mutations at amino acid positions 96, 104, 181, 304, 350 and
385, using the following oligonucleotides respectively.
[0082] OLXU 27: 5'-CTGACATGTTCTCTGAAAGAACCTCAGAGG-3'; SEQ ID NO.
21
[0083] OLXU 28: 5'-GTGTCCTGAGAACTGATCATTGCACTGACA-3'; SEQ ID NO.
22
[0084] OLXU 29: 5'-ATAAACGGAAATCTGCTCAAATTGTGGTGT-3'; SEQ ID NO.
23
[0085] OLXU 30: 5'-ACCCTCAGTGCTCTGACAAAGTGTTCCCAG-3'; SEQ ID NO.
24
[0086] OLXU 31: 5'-ACTGTCTGTAGACTGGGAATAAAAAGGAGG-3'; SEQ ID NO.
25
[0087] OLXU32: 5'-TCCTCCCGTTCCCTGCAGGAAGAGGTG-3'. SEQ ID NO. 26
[0088] Mutagenesis with above mutagenic primers resulted in
conversions of Asn (AAC or AAT) to Gln (CAG) at respective sites.
The resulting mutant constructs were designated gp75g1, gp75g2,
gp75g3, gp75g4, gp75g5 and gp75g6. Mutants were screened and
identified by DNA sequencing.
[0089] Mouse L cell fibroblasts were transfected with plasmids
containing full-length or mutant gp75 cDNA and pSV2 neo using
calcium phosphate precipitation method. The transfectants were
selected for growth in medium containing 500 .mu.g/ml effective
concentration of antibiotic Geneticin (GIBCO BRL Life Technologies,
Grand Island, N.Y.). Individual transfectant clones were isolated
using cloning rings (Bellco, Vineland, N.J.) and screened for gp75
expression by immunofluorescence staining with mAb TA99.
[0090] We first investigated which potential N-linked glycosylation
sites were used by comparing the molecular mass difference of
mutant gp75 proteins to immature, glycosylated wild-type gp75.
Transfectants expressing different gp75 glycosylation mutants were
labeled with [.sup.35S] methionine for 15 min followed by
immunoprecipitation with mAb TA99. B16 melanoma cells and wild-type
gp75 transfectants produced a sharp 71 kDa band of gp75,
representing an immature form of gp75 with high mannose sugar
chains characteristic of ER processing. Among the glycosylation
mutants, only gp75g2 appeared as a 71 kDa band, while all others
produced a 68 kDa band. Because one high mannose oligosaccharide
chain corresponds to approximately 3 kDa of molecular mass, the
observed difference between the molecular mass of the mutant gp75
proteins and wild-type gp75 is consistent with the interpretation
that the 68 kDa mutant gp75 molecules contained one less
carbohydrate chain than the wild-type gp75. This, in turn, is a
direct result of the abolishment of one carbohydrate chain at the
particular potential glycosylation site. Thus, it is reasoned that
these sites (Asn positions 96, 181, 304, 350 and 385 which are
individually mutated in gp75g1, g3, g4, g5 and g6) are normally
used for glycosylation. In contrast, the mutation at Asn 104
(mutated in gp75g2) did not cause any alteration in molecular mass
between gp75g2 and wild-type gp75; it is most likely that this site
is normally not used for glycosylation.
[0091] To assess the individual roles of each N-glycan in the
stability and transport of mouse gp75, we performed pulse-chase
metabolic labeling with [.sup.35S]methionine followed by
immunoprecipitation and Endo H digestion on each mutant gp75
transfectant, and compared the data to that of wild-type mouse gp75
expressed in L cell transfectants. Newly synthesized wild-type gp75
appeared as a doublet of 70 kDa and 68 kDa bands in the
transfectants at the end of 15 pulse and after a subsequent 15 min,
or 30 min chase. Endo H digestion reduced the bands to 57 and 52
kDa core polypeptide bands, showing that before 30 min chase, newly
synthesized gp75 remained in the ER. Starting from after 30 min
chase, gp75 appeared as a mature 72-79 kDa band, which was
resistant to Endo H digestion, because Endo H digestion could not
remove all N-glycans to the predicted core peptide size. This
indicates movement of gp75 protein from the ER or cis-Golgi (until
30 min chase) to the medial- or trans-Golgi 30 min after de novo
synthesis and further processing on the carbohydrate chains in
these compartment. The 72-79 kDa gp75 protein did not change
further after subsequent chase, indicating completion of
glycosylation on gp75. The mature gp75 is presumably further
transported to the endosomes/lysosomes, although the time course is
not reflected from this experiment. The intensity of the 72-79 kDa
band remained stable until 4 h after chase, indicating a half-life
of 4-8 h of the mature protein.
[0092] The above pulse-chase experiment followed by
immunoprecipitation with mAb TA99 and Endo H digestion reflecting
the intracellular stability as well as protein transport from the
ER to the Golgi was performed on all of the glycosylation mutants.
The cellular transport and stability of all the glycosylation
mutants can be grouped into 3 categories. (1) gp75g1 and gp75g3
appeared to have very similar transport pattern and stability data
as that of the wild-type gp75; (2) gp75g4 and gp75g6 proteins
remained Endo H sensitive with half-lives between 1-4 h, suggesting
retention and degradation in the ER; (3) gp75g5 was transported
from the ER to the Golgi in a similar rate as the wild type gp75,
yet displayed a shorter half life. At the end of 15 min pulse
labeling and 30 min chase, gp75g5 appeared as a 68 kDa which was
sensitive to Endo H to yield a 57 kDa band. (The additional 66 kDa
band with a core polypeptide of 52 kDa is a truncated form similar
to that in full length transfectants). At the end of 1 h chase,
majority of gp75g5 was converted to an Endo H resistant 75 kDa
band, showing that it was transported and processed to the medial-
or trans-Golgi, and the rate of transport was similar to that in
wild-type transfectants. Unlike that of the wild-type gp75, the
intensity of the 75 kDa band decreased after 1 h of chase. This
suggested that the half life of gp75g5 was between 1 to 4 h,
shorter than wild type gp75 in transfectants (T1/2=4-8 h).
Apparently, the abolishment of the N-linked carbohydrate chain at
position 350 affected the stability of the protein.
[0093] The above data showed that gp75g5 had a shorter half life
than wild-type gp75. It appeared to be transported to the Golgi,
and presumably further to the endosomes/lysosomes, as the transport
signal in the cytoplasmic tail is intact. In order to examine
whether gp75g5 was actually transported to the endosomes/lysosomes
and the shorter half-life of the protein was due to
endosomal/lysosomal degradation, we repeated the pulse-chase
experiment in the presence of NH.sub.4Cl, a lysosomotrophic weak
amine which inhibits proteases in acidic environments such as
endosomes or lysosomes, or leupeptin, a serine/cysteine protease
inhibitor which inhibits mainly proteases in the lysosomes. At the
end of 15 min label, the 68 kDa mutant gp75 band was synthesized
with equal intensity in the absence or presence of NH.sub.4Cl
incubation. With the absence of NH.sub.4Cl, the intensity of gp75g5
band reduced markedly at 4 h chase comparing with that at 0.5 h
chase. However, in the presence of NH.sub.4Cl, the gp75g5 band was
nearly as strong at the end of 4 h chase as at the end of 0.5 h
chase or after 15 min labeling. This result clearly showed a
prolonged half-life to more than 4 h for gp75g5 in the presence of
NH.sub.4Cl, and suggested that the short half life of gp75g5 was
due to rapid degradation in acidic compartments sensitive to
NH.sub.4Cl inhibition, which are most likely the late endosomes or
lysosomes. Similarly, in the presence of leupeptin, the intensity
of the gp75g5 band remained as the same after 4 h chase as after
0.5 h chase, showing a great stabilization of the protein. Based on
these results, it is concluded that gp75g5 was transported to the
endosomes/lysosomes, and was rapidly degraded there.
[0094] The above conclusion is also confirmed by immunofluorescence
staining of transfectants expressing gp75g5 in the absence and
presence of leupeptin. In wild-type gp75 transfectants, gp75 is
localized in the juxtanuclear patches and peripheral punctate
vesicles, which represent the Golgi complex and the late
endosomes/lysosomes. The juxtanuclear patches represented Golgi
apparatus, and the localization of wild-type full-length gp75 in
the Golgi apparatus at steady state suggested accumulation and slow
passage of gp75 in this compartment during transport. The staining
of gp75g5 transfectants showed only intensive juxtanuclear
structures, with no visible peripheral vesicles. The lack of
staining of peripheral vesicles indicated that at steady state,
there was no detectable level of gp75g5 in the endosomes and
lysosomes. However, staining of gp75g5 transfectants in the
presence of leupeptin revealed an enhanced overall staining of
gp75g5 transfectants, particularly, the peripheral vesicles became
visible. These peripheral vesicles are most likely endosomes and
lysosomes based on studies on the location of wild-type gp75 in the
transfectants. This result supports the above notion that leupeptin
stabilized the gp75g5 mutant proteins in endosomes and
lysosomes.
[0095] Taken together, the above data showed that the mutation at
Asn 350 to eliminate an oligosaccharide chain at this position
produced a mutant gp75 protein, which is more prone to proteolytic
digestions in the lysosomes than the wild-type gp75, and serine or
cysteine proteases were involved in the degradation process. This
mutation did not alter the route of intracellular sorting and
trafficking of gp75, as gp75g5 was still sorted to the
endosomes/lysosomes.
[0096] Pulse-chase experiments of gp75g1 and gp75g3 showed very
similar pattern of gp75 transport and stability compared to the
wild-type gp75. This result indicated that the N-glycans at Asn 96
and 181 (eliminated at gp75g1 and g3) are not involved in determine
the stability of gp75. Under immunofluorescence staining, gp75g1
was localized to juxtanuclear structure and peripheral vesicles,
just like the localization of wild-type gp75. Staining gp75g3
revealed predominantly perinuclear vesicles with non-visible
juxtanuclear patches, suggesting localization of gp75g3 mainly in
the endosomes and lysosomes at steady state. Since the juxtanuclear
patches are most probably the Golgi complex or early endosomes,
this result suggested an increased rate of transport of gp75g5
through the Golgi complex and the early endosomes than that of
wild-type gp75. Thus, N-glycan at Asn 181 seems to be involved in
the rate of transport through the Golgi.
[0097] Pulse-chase experiments of gp75g4 and gp75g6 showed a
different pattern of cellular transport and stability from that of
wild-type gp75 or other glycosylation mutants. After 15 min pulse
and after up to 4 h chase, gp75g4 and gp75g6 mutant proteins
remained to be 68 kDa, sensitive to Endo H digestion; and their
intensities decreased between 1 to 4 h of chase. These data
suggested ER retention and degradation of the mutant proteins.
Under immunofluorescence staining with mAb TA99, gp75g4 showed a
pattern of weak, diffuse staining mainly in fine perinuclear
networks, indicative of the ER network; while gp75g6 was mainly
localized in condensed perinuclear patches, which was consistent to
be the Golgi apparatus. Combining the biochemical and staining
data, it appears that gp75g4 is retained in the ER and gp75g6 is
retained mostly in the cis-Golgi apparatus. Thus, the elimination
of N-glycan at Asn 304 or Asn 385 affected the cellular transport
of the protein from the ER to the Golgi. Malfolding may be the
mechanism for the retention as suggested by a lot of earlier
studies.
* * * * *