Patent Application: US-26713702-A

Abstract:
this invention relates generally to the field of growth factor . in particular , the invention provides an isolated nucleic acid fragment , comprising a sequence of nucleotides encoding a kringle domain 1 of human hepatocyte growth factor . proteins or peptides encoded by the hgfk1 nucleic acids are also provided . compositions comprising hgfk1 nucleic acids , proteins , or functional derivatives or fragments are also provided . methods for producing and / or using hgfk1 nucleic acids , proteins , or functional derivatives or fragments are further provided .

Description:
for clarity of disclosure , and not by way of limitation , the detailed description of the invention is divided into the subsections that follow . unless defined otherwise , all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs . all patents , published patent applications and other publications and sequences from genbank and other databases referred to herein are incorporated by reference in their entirety . if a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in patents , published patent applications and other publications and sequences from genbank and other data bases that are herein incorporated by reference , the definition set forth in this section prevails over the definition that is incorporated herein by reference . as used herein , “ a ” or “ an ” means “ at least one ” or “ one or more .” as used herein , “ kringle domain 1 of human hepatocyte growth factor ( hgfk1 )” includes those variants with conservative amino acid substitutions that do not substantially alter their cell growth or angiogenesis inhibiting activity . suitable conservative substitutions of amino acids are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule . those of skill in this art recognize that , in general , single amino acid substitutions in non - essential regions of a polypeptide do not substantially alter biological activity ( see , e . g ., watson et al . molecular biology of the gene , 4th edition , 1987 , the bejacmin / cummings pub . co ., p . 224 ). as used herein , a “ functional derivative or fragment of hgfk1 ” refers to a derivative or fragment of hgfk1 that still substantially retains its function as a pathological cell growth or angiogenesis inhibitor . normally , the derivative or fragment retains at least 50 % of its pathological cell growth or angiogenesis inhibiting activity . preferably , the derivative or fragment retains at least 60 %, 70 %, 80 %, 90 %, 95 %, 99 % and 100 % of its pathological cell growth or angiogenesis inhibiting activity . as used herein , an “ agent that enhances cell growth or angiogenesis inhibiting function of hgfk1 ” refers to a substance that increases potency of hgfk1 &# 39 ; s pathological cell growth or angiogenesis inhibiting activity , or a substance that increases sensitivity of a hgfk1 &# 39 ; s natural ligand in an pathological cell growth or angiogenesis inhibiting signaling pathway , or a substance that decreases potency of a hgfk1 &# 39 ; s antagonist . as used herein , an “ agent that enhances production of hgfk1 ” refers to a substance that increases transcription and / or translation of a hgfk1 gene , or a substance that increases post - translational modification and / or cellular trafficking of a hgfk1 precursor , or a substance that prolongs half - life of a hgfk1 protein . as used herein , a “ combination ” refers to any association between two or among more items . as used herein , a “ composition ” refers to a any mixture of two or more products or compounds . it may be a solution , a suspension , liquid , powder , a paste , aqueous , non - aqueous or any combination thereof . as used herein , “ nucleic acid ” refers to any nucleic acid containing molecule including , but not limited to dna , rna or pna . the term encompasses sequences that include any of the known base analogs of dna and rna including , but not limited to , 4 - acetylcytosine , 8 - hydroxy - n6 - methyladenosine , aziridinylcytosine , pseudoisocytosine , 5 -( carboxyhydroxylmethyl ) uracil , 5 - fluorouracil , 5 - bromouracil , 5 - carboxymethylaminomethyl - 2 - thiouracil , 5 - carboxymethylaminomethyluracil , dihydrouracil , inosine , n6 - isopentenyladenine , 1 - methyladenine , 1 - methylpseudouracil , 1 - methylguanine , 1 - methylinosine , 2 , 2 - dimethylguanine , 2 - methyladenine , 2 - methylguanine , 3 - methylcytosine , 5 - methylcytosine , n6 - methyladenine , 7 - methylguanine , 5 - methylaminomethyluracil , 5 - methoxyaminomethyl - 2 - thiouracil , beta - d - mannosylqueosine , 5 ′- methoxycarbonylmethyluracil , 5 - methoxyuracil , 2 - methylthio - n6 - isopentenyladenine , uracil - 5 - oxyacetic acid methylester , uracil - 5 - oxyacetic acid , oxybutoxosine , pseudouracil , queosine , 2 - thiocytosine , 5 - methyl - 2 - thiouracil , 2 - thiouracil , 4 - thiouracil , 5 - methyluracil , n - uracil - 5 - oxyacetic acid methylester , uracil - 5 - oxyacetic acid , pseudouracil , queosine , 2 - thiocytosine , and 2 , 6 - diaminopurine . as used herein : “ stringency of hybridization ” in determining percentage mismatch is as follows : 1 ) high stringency : 0 . 1 × sspe , 0 . 1 % sds , 65 ° c . ; 2 ) medium stringency : 0 . 2 × sspe , 0 . 1 % sds , 50 ° c . ( also referred to as moderate stringency ); and 3 ) low stringency : 1 . 0 × sspe , 0 . 1 % sds , 50 ° c . it is understood that equivalent stringencies may be achieved using alternative buffers , salts and temperatures . for clarity of disclosure , and not by way of limitation , the detailed description of the invention is divided into the subsections that follow . the following example is included for illustrative purposes only and is not intended to limit the scope of the invention . kringle 1 of human hepatocyte growth factor inhibits bovine aortic endothelial cell proliferation stimulated by basic fibroblast growth factor and causes cell apoptosis reagents and materials . dmem and trypsin / edta were purchased from gibco brl ( rockville , md .). fetal calf serum were from hyclone ( logen utah ). human fresh placenta was from shanghai no . 1 women and children healthcare hospital . angiostatin was prepared in our lab as described previously ( 9 ). rt - pcr . human fresh placenta was homogenized in liquid nitrogen . total rna was isolated using trizol reagent ( gibco brl ). total rna was used as the template for cdna synthesis using superscript ™ rnase h − transcriptase ( gibco brl ) according to manufacturer &# 39 ; s instructions . pcr was performed with ex - taq dna polymarase ( takara ) according to manufacturer &# 39 ; s instructions . the synthetic oligonucleotides were obtained from shanghai sangon co . ltd . ( shanghai china ). the primers used were as follows : rt primer : 5 ′ gcaggtcatgcattcaac 3 ′ ( seq id no : 4 ), primers used for amplifying cdna encoding hgfk1 , sense primer : 5 ′ ggaattccatatgaactgcatcattggtaaagga 3 ′ ( seq id no : 5 ), antisense primer : 5 ′ atcgaa gcttattaatggtggtgatggtggtggcaggtcatgcattc ( seq id no : 6 ), a ndei site was included in the sense primer , a hindiii site , a stop codon and six histidine codon were incorporated into antisense primer . pcr product of 290 bp was amplified with this primer sets . reaction were incubated in pe480 thermal cycler ( perkin - elmers , nj ) for 35 cycles : denaturation 30 s , 94 ° c . ; annealing 30 s , 52 ° c . ; extension 30 s , 72 ° c . pcr product was run on 1 % agarose gel in tbe ( 10 mm tris - borate , 1 mm edta , ph 8 . 0 ), and visualized by ethidium bromide staining . gene construction and expression . the amplified cdna fragment was ligated into the ndei / hindiii sites of escherichia coli expression vector pet24 - a ( novagen ), resulting in the expression plasmid pethk1 . pethk1 was transformed into e . coli bl21 ( de3 ) and hgfk1 expression was induced by 1 mm iptg . cells were harvested by centrifugation for 30 min at 4000 g . purification and refolding of recombinant hgfk1 . cells were resuspended in 20 mm tris - hcl , ph 8 . 0 , 50 mm kcl , 0 . 5 mm edta , 5 mm dte and lysozyme was added to the final concentration of 0 . 5 mg / ml . cells were incubated at 4 ° c . for 30 min , then were disrupted by sonic homogenizer for 10 sec for six times with 30 sec interval each time . after centrifugation at 4 ° c ., 12 , 000 g for 30 min , the pellet was collected and resuspended in 8 m urea , 0 . 1 m nah 2 po 4 , 10 mm tris - hcl , ph 8 . 0 . centrifuged again as before , the supernatant was loaded on a ni 2 + - nitrilotriacetic acid - agarose column ( qiagen ). the recombinant protein was eluted from the column according to manufacturer &# 39 ; s instructions . to achieve refolding , the purified protein were adjusted to ph 8 . 0 and dtt was added to the final concentration of 0 . 1 m . following incubation at room temperature for 2 hours , the solution was added to refolding buffer ( 0 . 1 m tris - hcl , ph 8 . 0 , 0 . 5 m arginine , 5 mm edta , 1 mm gssg , 5 mm gsh ) with the ratio of 1 : 200 ( v / v ). after 24 hours &# 39 ; incubation at room temperature , the renatured protein was dialyzed against distilled water for 24 - 48 hours and lyophilized . bovine aortic endothelial cell proliferation assay . bovine aortic endothelial cell were isolated as previously described ( 10 ) and were maintained in dmem supplemented with 10 % heat - inactivated fcs and antibiotics . monolayer of bae cells growing in 60 mm dish were dispersed in 0 . 05 % trypsin solution . cells were resuspended with dmem containing 10 % fcs . approximately 3000 cells in 200 ul were added in triplicate to each well of 96 - well tissue culture plates and incubated at 37 ° c . ( in 10 % co 2 ). cells adhere to the plate in about 2 - 3 hours . the medium was replaced with 100 ul of fresh dmem containing 2 % fcs , and samples of hgfk1 or angiostatin were added to each well . after 30 minutes &# 39 ; incubation , another 100 ul dmem containing 2 % fcs and 10 ng / ml bfgf was added to each well . after 72 hours &# 39 ; incubation , 10 ul mtt ( 100 mg / ml ) was added to each well and incubated for another 4 hours at 37 ° c ., 10 % co 2 . 180 ul medium was pipetted out from each well and 50 ul dmso was added , vortex gently to dissolve the pellet . the absorbance of a 570 nm , which correlates to the number of cells , was measured with microplate reader ( model 450 , bio - rad ). flow cytometry apoptosis analysis by propidium iodide ( pi ) assay . all the procedures were followed as previously reported ( 11 ). briefly , bae cells were maintained in dmem supplemented with 10 % fcs till to 60 - 70 % confluence . the medium was changed with dmem supplemented with 0 . 5 % fcs containing 2 ug / ml hgfk1 . an hour later , bfgf was added to the final concentration of 5 ng / ml and cells were cultured overnight at 37 ° c . in 10 % co 2 . cells were trypsinized and washed gently with pbs , and then were fixed with 70 % ice - cold ethanol for 30 minutes . cells were collected by centrifugation . 200 ul 1 mg / ml rnase was added and incubated at 37 ° c . for 30 minutes , then 400 ul pi ( 500 ug / ml ) was added and incubated dark at 4 ° c . for 30 minutes . cells were assessed by facstar plus flow cytometer ( beckton - dickinson ) for apoptosis and the results were analyzed with cellquest software . purification and characterization of hgfk1 . recombinant protein including amino acids residues from 127 - 214 of hgf plus six histidines was expressed in e . coli . and purified to homogeneity using ni 2 + - nitrilotriacetic acid - agarose column and was refolded in vitro ( fig1 ). under reducing condition , hgfk1 migrated in page with molecular mass of about 11 kd , corresponding to the predicted molecular mass . inhibition of bae cell proliferation with recombinant hgfk1 and human angiostatin . hgfk1 and angiostatin were assayed for their inhibitory activities on bovine aortic endothelial cell growth stimulated by bfgf . as shown in fig2 both angiostatin and hgfk1 inhibited bae cell proliferation in a dose - dependent fashion . the concentration of hgfk1 required to reach 50 % inhibition ( ed50 ) was about 0 . 7 ug / ml , while ed50 of angiostatin is 3 ug / ml , close to data previously reported ( 4 ). while both hgfk1 and angiostatin have no inhibitory activities on fibroblast cell line balb / c3t3 and hepatoma cell line hepg2 ( data not shown ), which suggests that their inhibitory activity is specific to endothelial cell . cell apoptosis detection . as shown in fig3 bae cells were treated with 2 ug / ml hgfk1 overnight in 0 . 5 % fcs , about 23 % cells underwent apoptosis , compared with 3 % of untreated cells . kringle domains were found in many proteins , most of which are important molecules mediating coagulation and fibrinolysis or lipid transportation . therefore , kringle modules were thought to function as recognition unit for protein and protein or protein and cell surface ( 1 ). however , in recent years , several proteins containing kringle module were found to inhibit specifically the proliferation of endothelial cell . angiostatin , which is composed by the first four kringles of plasminogen was found to inhibit endothelial cell proliferation stimulated by bfgf ( 9 ) and except kringle 4 , each kringle of plasminogen had more or less anti - angiogenesis activity ( 5 ). lee et al . ( 6 ) reported that kringle 2 of prothrombin had inhibitory activity against bce proliferation . reports about apolipoprotein a were contradictory . lou et al . ( 7 ) found that it displayed no such kind of activity despite that its kringle showed great sequence similarity with kringle 4 of plasminogen ; while trieu et al . ( 8 ) reported that recombinant apolipoprotein a with 18 kringle 4 repeats impaired angiogenesis in animal model experiment . these reports , combined together , implied that some kringle domains manifested anti - angiogenic activity . amino acid sequence alignment of the kringle domains of plasminogen and hgfk1 showed that they displayed considerable similarity ( 40 - 50 % identity , table 1 ). we thus wondered whether hgfk1 was also an angiogenesis inhibitor . our result in this paper demonstrated for the first time that hgfk1 is indeed an angiogenesis inhibitor . in the examples above , the “ mother ” proteins display no inhibitory activity on angiogenesis and hgf is even a growth factor with angiogenic activity . therefore , we proposed that in “ mother ” proteins the functional elements of kringle were shielded and could not interact with endothelial cell effectively . while after kringle domains were set free from “ mother ” molecules , they were more freely to access the binding sites on endothelial cell membrane and manifested anti - angiogenic function . this phenomenon might be regarded as the result of the diversity of protein function in molecule evolution . then , why different kringles inhibited the proliferation of endothelial cell in different degree ? we contributed it to their subtle differences in amino acids sequence , as well as in their second structure . it has been reported that lysine - binding site in kringle was not related with inhibitory activity ( 4 ); the treatment of kringle structure by reductive reagent may compromise its inhibitory activity ( 4 ); kringle 4 is distinctive among kringles of plasminogen for not having such inhibitory activity . cao et al . ( 5 ) contributes it to two sets of consecutive lysine residues in its amino acids sequence . therefore , much effort is needed to locate the exact amino acids clusters that count most . as was seen in fig2 hgfk1 was 3 times stronger than angiostatin in inhibiting the proliferation of endothelial cell . we believe that hgfk1 might share the same receptor with angiostatin on cell membrane with different kd , which led to the different degree of their inhibitory activity . moser et al reported that atp synthase on the membrane of huvec cell line was the receptor of angiostatin ( 12 ). they suggested that angiostatin bound to atp synthase and rendered endothelial cells more vulnerable to hypoxic challenge and eventual irreversible cell damage ( 12 ). however , direct evidence has not been reported as to whether binding of atp synthase to angiostatin could block its enzymatic activity or not . angiostatin was reported to induce endothelial cell apoptosis ( 13 ). we here demonstrated that hgfk1 could induce bae cell apoptosis , too . little is known about their mechanism . liu et al . reported that angiostatin treatment of ecv304 , a well characterized human umbilical endothelial cell line , could block vegf or bfgf induced down regulation of caveolin - 1 ( 14 ). caveolin - 1 is the marker protein of caveolae , whose function was regarded to be transportation of materials from outside to inside cell membrane ( 15 ). atp synthase was found in caveolae ( 16 ), which made us suspect that kringles in angiostatin and hgfk1 may inhibit cell proliferation stimulated by bfgf and induce cell apoptosis at least partly by disturbing material transportation through caveolae on cell membrane . kringle 1 of human hepatocyte growth factor differentially regulates caveolin - 1 expression in bovine aortic endothelial cell kringle domain is a kind of protein module , which usually consists of about 80 amino acids ( 1 ). many proteins with this domain , for example , angiostatin and hgfk1 , are reported to manifest anti - angiogenic activity ( 2 )( 5 ). we previously reported that hgfk1 inhibited specifically the proliferation of bovine aortic endothelial cell stimulated by bfgf , with ed 50 of about 0 . 7 ug / ml and caused bae cell apoptosis ( 17 ). however , the exact mechanism by which it performs its activity is not clear yet . caveolin - 1 is a membrane protein abundantly found in caveolae , the vesicular invaginations of plasma membrane ( 18 ). its topology is unusual in that both its n - terminal and c - terminal domains face cytoplasm ( 15 ). many important molecules , including g protein coupled receptors , receptor tyrosine kinase , are found to bind with caveolin in caveolae ( 15 ). direct interaction of caveolin with signaling molecules leads to their inactivation ( 15 ). liu et al reported that bfgf induced down - regulation of caveolin - 1 in ecv304 , a well characterized endothelial cell line , and that angiostatin could block this down - regulation ( 14 ). in this example , we demonstrated for the first time that hgfk1 could block the down - regulation of caveolin - 1 induced by bfgf in bae cell line . therefore , all the proteins with kringle domain might display their inhibitory activity via same mechanism in endothelial cells . reagents and materials . dmem and trypsin / edta were purchased from gibco brl ( rockville , md .). fetal calf serum were from hyclone ( logen utah ). polyclonal anti - caveolin - 1 igg was from santa cruz biotechnologies , inc . ( santa cruz , calif .). angiostatin and hgfk1 were prepared in our lab as described previously ( 17 )( 9 ). cell culture and treatment with angiostatin and hgfk1 . bovine aortic endothelial cells were isolated as previously described ( 10 ) and were seeded at a density of ˜ 1 . 0 × 10 4 cells / ml in 24 - well plates , maintained in dmem supplemented with 10 % heat - inactivated fcs and antibiotics . after incubation in normal growth medium overnight , the medium was replaced by dmem containing 5 % fetal calf serum with or without bfgf , angiostatin or hgfk1 at different concentrations according to the purpose of the assay . protein analysis . expression of caveolin - 1 was examined by western blot analysis . cells were solubilized with sample buffer containing 0 . 125 m tris - hcl ( ph 6 . 8 ), 5 % ( w / v ) sds , 2 . 5 %( v / v ) β - mercaptoethanol , 5 % glycerol in double distilled water . after boiling for 4 min , proteins were separated by sds - polyacrylamide gel electrophoresis ( 5 - 15 % gradient gel ), transferred to nitrocelloses , and subjected to western blot analysis using enhanced chemilunescence . prior to loading , the protein concentration of the samples was measured with the bicinchoninic acid method using bovine serum albumin as a standard . angiostatin and hgfk1 cause morphological changes in bae cell . normally , bovine aortic endothelial cells adhere tightly to the cell dish as shown in fig4 ( a ). however , after exposure under 5 ug / ml angiostatin or 2 ug / ml hgfk1 for 24 hours , distinct morphological changes were observed . as were shown in fig4 ( b ) and ( c ), some of the cells retracted from the cell dish and turned round morphologically under microscope . hgfk1 differentially regulates caveolin - 1 expression in bae cell . bfgf was reported to down - regulate the expression of caveolin - 1 protein in ecv 304 cell line . here we demonstrated that bfgf had the same effect on bae cell line . we determined the time and concentration dependence of the effects of bfgf . as was shown in fig5 ( a ), bfgf induced the down - regulation of caveolin - 1 at a minimal concentration of 3 ng / ml , and the effect became maximal at 10 ng / ml . bfgf - induced down - regulation of caveolin - 1 appeared after 3 h of treatment , exerting its maximal effects at 24 h . ( fig5 b ). the down - regulation of caveolin - l by bfgf was blocked by 5 ug / ml angiostatin or 2 ug / ml hgfk1 ( fig6 ). seven years have passed since the first publication of angiostatin and the related kringle domains . after that , a large number of literatures have been published to help advance their practicality in clinical research . however , few proceeding is accomplished as to the exact mechanism , by which they perform their activities . in this example , we try to help throw some light in this area . up till now , there is only one published explanation as to how angiostatin inhibits the metastasis of endothelial cell : kinetic analysis demonstrated that angiostatin functions as a non - competitive inhibitor of extracellular - matrix - enhanced , tpa - catalysed plasminogen activator ( 19 ); while there are two kinds of mechanisms to interpret how angiostatin and molecules alike inhibit the proliferation of endothelial cell . first , moser et al reported that angiostatin bound to atp synthase on huvec membrane . they speculated that the plasma membrane associated atp synthase may produce extracellular atp , which can diffuse back into the cell , providing an additional , albeit limited , atp source . binding of angiostatin with atp synthase disrupted the production of atp and rendered endothelial cell more vulnerable to hypoxic challenge and eventual irreversible damage ( 12 ). second , liu et al demonstrated that angiostatin could block the down - regulation of caveolin - 1 in ecv304 cell line and thus affect the mapk signaling pathway ( 14 ). in this example , we demonstrated for the first time that hgfk1 and angiostatin could block the down - regulation of caveolin - 1 in bae cell line . therefore , we assume that all the proteins with kringle domain might perform their activities through the same mechanism in endothelial cells . it is worthwhile to mention that atp synthase is found in caveolae ( 16 ), therefore , it is very interesting to propose , as is shown in fig7 that , besides the stimulation with bfgf , the dissociation of caveolin - 1 and signaling molecules in caveolae might need energy , which could be provided by atp synthase nearby in caveolae . after the dissociation , caveolin - 1 is degraded via endosome pathway , so , once the cells are stimulated with bfgf , caveolin - 1 is down regulated . however , if hgfk1 or angiostatin is added to the cell culture medium , it binds to atp synthase and blocks the production of atp , which makes the dissociation of caveolin - 1 and the signaling molecules impossible . in that case , cells fail to proliferate and the caveolin - 1 is not down regulated , which is compatible with our result . 1 . castellino , f . j . and mccance , s . the kringle domains of human plasminogen . from plasminogen - related growth factors . 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( 1998 ) proc . natl . acad . sci . usa 95 , 5579 - 5583 . 14 . liu , j ., razani , b ., tang s . q ., terman , b . i ., ware , a . and lisanti , m . p . ( 1999 ) j . biol . chem . 274 ( 22 ), 15781 - 15785 . 15 . okamoto , t ., schlegel , a ., scherer , p . e . and lisandi , m . p . ( 1998 ) j . biol . chem . 273 ( 10 ), 5419 - 5422 . 17 . xin l , xu r , zhang q , li z . p ., gan r . b . ( 2000 ) biochem . biophy . res . communn . 277 ( 1 ) 186 - 190 . 18 . sargiacomo m , scherer p . e ., tang z , kubler e , song k . s ., sanders m . c ., lisanti m . p . ( 1995 ) proc . natl . acad . sci . u . s . a . 92 : 9407 - 9411 . 19 . stack m . s ., gately s , bafetti l . m ., enghild j . j ., soff g . a . ( 1999 ) biochem . j . 340 : 77 - 84 the above examples are included for illustrative purposes only and is not intended to limit the scope of the invention . since modifications will be apparent to those of skill in this art , it is intended that this invention be limited only by the scope of the appended claims .