John Connell Laboratory of Glycobiology, Austin Research Institute, Austin and Repatriation Medical Centre, Heidelberg Victoria 3084, Australia
Received on March 3, 2002; revised on May 27, 2002; accepted on June 13, 2002
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: -galactosidase/cell surface/furin/targeting/trans-Golgi network
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For pig-to-human xenotransplantation, the carbohydrate structure Gal(1,3)Gal expressed on glycoproteins and glycolipids at the cell surface of pig endothelium causes the rapid antibody-mediated rejection process, hyperacute rejection (HAR). If Gal
(1,3)Gal is modified or cleaved, HAR can be significantly reduced if not eliminated (Sandrin and McKenzie, 1999
). One enzyme of interest that can cleave Gal
(1,3)Gal is galdase. When this enzyme is overexpressed in the cell, it is capable of cleaving and hence reducing the amount of Gal
(1,3)Gal on the cell surface (Osman et al., 1997
).
Galdase hydrolyses terminal -Gal from glycoproteins and glycolipids. The enzyme is soluble, contains no transmembrane domain, and is localized to the lysosome of the cell. For galdase to hydrolyze Gal
(1,3)Gal, both the enzyme and the epitope must occupy the same cellular location. This location is almost certainly within the trans region of the Golgi, where newly synthesized galdase accumulates before its transport to the lysosome via a mannose-6-phosphate (M6P) signal at position N215 (Ioannou et al., 1998
). Exceptions to this are when galdase is overexpressed and swamps the M6P receptor pathway in the cell or when the M6P signal is removed by mutating N215 to A215; in both cases this results in exportation of the enzyme from the cell by a secretory pathway (Ioannou et al., 1992
, 1998). The trans region of the Golgi is also the site of Gal
(1,3)Gal synthesis as a terminal carbohydrate moiety on glycoproteins and glycolipids prior to their transport to the cell surface.
An effective reduction of Gal(1,3)Gal, could be achieved by relocalizing galdase to be in closer proximity to newly synthesized Gal
(1,3)Gal, by targeting galdase to: (1) the trans-Golgi network (TGN), the region of the Golgi in which exportation of glycoproteins destined for the cell surface occurs and therefore where effective cleavage of Gal
(1,3)Gal is possible prior to cell surface expression; or (2) the cell surface, where cleavage may occur while galdase and Gal
(1,3)Gal are transported to the cell surface or at their final destination on the cell surface. All of the Golgi resident proteins identified to date are either integral Golgi membrane proteins (including glycosyltransferases, such as fucosyltransferases, galactosyltransferases, and sialyltransferases), endoproteases (e.g., furin), or cytosolic Golgi membraneassociated transport proteins (such as COP I and II complexes, AP1 complex, clathrin). Targeting galdase, to the TGN and cell surface could be achieved by (1) attaching a transmembrane domain to tether the enzyme to the Golgi membrane or plasma membrane, and (2) attaching a cytoplasmic domain containing a TGN localization signal.
The TGN targeting domain chosen was the transmembrane and cytoplasmic domains from the Type I TGN resident protein furin, which is a human subtilisinlike protein. Extensive studies on the cytoplasmic domain shows that the majority of furin is found in the TGN (Bosshart et al., 1994; Jones et al., 1995
; Molloy et al., 1994
; Schafer et al., 1995
; Shapiro et al., 1997
) with a small proportion cycling to and from the plasma membrane (Bosshart et al., 1994
). The amino acid sequence of furin between P772 and R784 contains an acidic cluster and a casein kinase II consensus sequence that together retain furin in the TGN (Jones et al., 1995
; Schafer et al., 1995
). A tyrosine-based signal Y762KGL765 provides endosomal localization (Bosshart et al., 1994
) allowing the retrieval of cell surfaceexpressed furin to the TGN. Complete removal of these signals causes furin to be expressed only at the cell surface (Schafer et al., 1995
).
The use of carboxy-terminal sequences of furin has enabled the design of two chimeric galdase proteins: (1) galdase-furin(Tm+Cyto), containing the transmembrane region and cytoplasmic domains of furin, with the intention of localizing galdase to the TGN and allowing some cycling to the cell surface; (2) galdase-furin(Tm), containing the transmembrane domain of furin, with the intention of expressing galdase at the cell surface. We now show that using the transmembrane domain and localization signals of furin, galdase can indeed be localized to several sites within the cell. This relocalization of galdase resulted in a more effective decrease in terminal -Gal at the cell surface. Such a strategy can be applied to xenotransplantation to decrease the effects of HAR. Moreover, relocalizing galdase may also be useful in enzyme replacement therapy for patients suffering from Fabrys disease, which is caused by a deficiency in galdase activity.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Intracellular distribution of chimeric galdases
Confocal microscopy was used to detect the intracellular localization of the chimeric enzymes and to compare this distribution with galdase. Cells expressing galdase predominantly showed a punctate intracellular distribution (Figure 2A) characteristic of lysosomal localization, which contrasted with mock-transfected cells showing weak background staining (Figure 2B). Staining for -adaptin, a TGN-localized protein, gave perinuclear foci typical of the Golgi (Figure 2C, D). There was a small proportion of coincident perinuclear staining of galdase with
-adaptin (Figure 2E) indicating that some galdase is normally found in the Golgi. These observations may be explained by (1) galdase en route (via the M6P-mediated pathway) through the Golgi to the lysosome or (2) the overexpression of galdase, causing a swamping of the M6P pathway and is present transiently in the Golgi, while en route to the cell surface.
|
|
To further characterize the localization of galdase-furin(Tm+Cyto) the fungal metabolite brefeldin A (BFA) was used to treat cells. This drug causes the separation of Golgi and the TGN, with resorption of the Golgi into the ER and the cessation of vesicular transport to and from the cell surface. The TGN remains as a tight perinuclear focus close to the microtubule organizing center (Reaves and Banting, 1992). Following BFA treatment, galdase-furin(Tm+Cyto) was retained in the TGN (green, Figure 3H). Vesicular staining was markedly decreased, as would be the case if the staining was due to vesicular transport to or from the cell surface. This active localization of galdase-furin(Tm+Cyto) to the TGN confirms previous reports for the colocalization of furin with TGN38 (Schafer et al., 1995
), another TGN marker. The distribution of the marker
-adaptin, however, was dispersed following treatment with BFA (red, Figure 3H) due to BFA blocking its recruitment to the Golgis cytosolic surface (Molloy et al., 1994
). These results demonstrate that galdase can be relocalized in the cell to the Golgi apparatus, specifically the TGN, by attaching the TGN-targeting signals contained within the cytoplasmic domain of furin.
As expected, galdase-furin(Tm) was found to localize to the cell surface either with or without cycloheximide treatment (Figure 4A, B), which was in contrast to nonchimeric galdase and galdase-furin(Tm+Cyto), where neither enzyme was observed at the cell surface (data not shown). Permeabilized galdase-furin(Tm)expressing cells (Figure 4C) showed a distribution pattern similar to galdase-furin(Tm+Cyto) with a small amount of perinuclear staining that colocalized with -adaptin (Figure 4E, Figure 3C). This staining of galdase-furin(Tm) was completely abrogated by cycloheximide treatment (Figure 4D), and only the
-adaptin staining was retained (Figure 4F). Thus no superimposable staining of
-adaptin and galdase-furin(Tm) was present (Figure 4H). This was the reverse to what was observed for galdase-furin(Tm+Cyto) following the same treatment (Figure 3G). Interestingly, the small amount of vesicular staining for galdase-furin(Tm) was also reduced by cycloheximide, indicating that this was a transient state of the enzyme as it is trafficked via vesicular transport from the TGN to the cell surface. The results indicate that galdase-furin(Tm), lacking a TGN signal, contains no means of being retained in the TGN and is instead trafficked to the cell surface. This is in agreement with previous findings for furin when the cytoplasmic tail signals were deleted (Schafer et al., 1995
).
|
|
|
To confirm that the observed decrease in Gal(1,3)Gal was indeed the result of cleavage by the galdase chimeras, NAcLac expression was examined by EcorA-FITC binding. The cleavage of Gal
(1,3)Gal by galdase produces NAcLac. EcorA-FITC binding increased for galdase-furin(Tm+Cyto) (Figure 6C); similarly, galdase and galdase-furin(Tm) cotransfected CHOP cells showed increases (data not shown). This was not due to the transfection of
(1,3)GT alone, which gave a minor decrease in EcorA-FITC fluorescence (data not shown), nor the transfection of the galdases without
(1,3)GT, which caused neither an increase nor decrease (data not shown) confirming that in the cotransfections the decrease in Gal
(1,3)Gal on the cell surface is the sole action of the expression of galdase and its enzymatic cleavage.
Both COS and CHOP cells are transient expression systems that do not express Gal(1,3)Gal on their cell surface. To see if a similar trend occurs in a cell line that expresses Gal
(1,3)Gal, the pig endothelial cell line (PIEC) was used. Two representative clones, one expressing galdase and one expressing galdase-furin(Tm+Cyto) were selected for further analysis. Both have comparable enzyme activities of approximately 140 nmoles/mg protein/h (Figure 7A) that are significantly elevated compared to the parental PIEC, 60 nmoles/mg protein/h (Figure 7A). Both gave a similar reduction in Gal
(1,3)Gal of ~50% compared with parental PIEC (Figure 7B, C), which was a greater reduction than was achieved in the transient expression systems (see previous discussion).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Galdase-furin(Tm+Cyto) localized to the TGN, as shown by colocalization studies with TGN markers -adaptin and WGA (Figure 3), indicating that the addition of the furin cytoplasmic domain sequence is sufficient for TGN localization. The results are similar to previous studies with furin using the TGN markers
-adaptin (Molloy et al., 1994
) and TGN38 (Molloy et al., 1994
; Schafer et al., 1995
; Shapiro et al., 1997
) and to immunoelectron microscopic studies (Bosshart et al., 1994
; Molloy et al., 1994
; Schafer et al., 1995
). Furthermore, findings have demonstrated a direct association of the furin cytoplasmic domain with the AP1 complex (Teuchert et al., 1999
), of which
-adaptin is a subunit, strengthening the evidence for TGN colocalization of
-adaptin and galdase-furin(Tm+Cyto) (Figure 3). However, it cannot be discounted that galdase-furin(Tm+Cyto) may be influenced by the lysosomal M6P signal contained within its galdase domain (Ioannou et al., 1998
), although confocal microscopy did show lower vesicular staining than that observed for galdase (Figure 2A, 3A). Also, BFA was able to reduce the vesicular staining significantly, indicating that it was probably trafficking to and from the cell surface via an endosomal pathway rather than localizing to the lysosomes. Another important observation was that the cytoplasmic domain sequence of furin was capable of cycling galdase-furin(Tm+Cyto) between the cell surface and the TGN (Figure 5D).
The CD7 stalk region was included in the galdase-furin(Tm+Cyto) construct as a spacer, spacing galdase from the lipid bilayer. The CD7 stalk is an extended structure containing four repeats with a high content of P, S, and T residues and provides a spacer between the transmembrane and Ig-like domains of CD7 (Aruffo and Seed, 1987). There are no localization signals in the stalk region, and even if such signals did exist they would be isolated from interacting with cytosolic transport proteins by the lipid bilayer, being positioned on the lumenal side of the bilayer. It is therefore unlikely that the CD7 stalk region included in the galdase-furin(Tm+Cyto) chimera contributed to its localization.
Deletion of the furin localization signals causes a dramatic change in its distribution in the cell such that it localizes to the plasma membrane and primary vesicles of the endosomal/lysosomal system and is unable to be retrieved from the cell surface (Bosshart et al., 1994; Schafer et al., 1995
). Galdase-furin(Tm) localization is in agreement with these observations, that is, galdase-furin(Tm) was found at the plasma membrane (Figure 4A, B) and was unable to cycle from the cell surface to the TGN (Figure 5E). Cycloheximide treatment of galdase-furin(Tm) expressing COS cells (Figure 4G, H) demonstrated two interesting observations. First, any galdase-furin(Tm) localized to the Golgi was due to its trafficking to the cell surface (most cell surface proteins follow this pathway); second, the lack of lysosomal staining demonstrated an inability of galdase-furin(Tm) to localize to the lysosome. The transmembrane domain from furin is sufficient to override the M6P signal of the galdase domain and this could be caused by the transmembrane domain anchoring galdase-furin(Tm) into the lipid bilayer, stopping either the synthesis of the M6P signal via N215 or its recognition.
The enzyme activity of the chimeric galdases was not compromised by the addition of furin transmembrane or cytoplasmic domain sequences. Indeed, there was increased galdase activity, which can be explained by a greater retention of active membrane-bound enzyme within the cell. In addition, the ability of the chimeric galdases to cleave Gal(1,3)Gal demonstrates that an increase in enzyme activity does indeed correspond to a greater decrease in cell surface Gal
(1,3)Gal (Figure 6A, B). However, relocalization of galdase gave only a modest improvement in the reduction of cell surface Gal
(1,3)Gal for transfected cell lines (Figure 6B). When expressed as a transgene in mice, a more significant reduction was observed for splenocytes, with galdase-furin(Tm+Cyto), 25%, compared to galdase, 0% (Figure 8A, D). A greater decrease in cell surface Gal
(1,3)Gal was not observed in the cell lines possibly as the overexpression of galdase caused the enzyme to follow a similar transport pathway as the chimeric galdases, from the trans region of the Golgi to the cell surface. Consequently, all three galdases (chimeric and nonchimeric) would be capable of cleaving Gal
(1,3)Gal along this pathway, masking the effectiveness observed for the chimeric galdases. Therefore when comparing chimeric and nonchimeric galdases, the expression level in the cell type concerned could dictate the degree of their effectiveness.
Targeting galdase is clearly a useful strategy to decrease cell surface Gal(1,3)Gal for pig-to-human xenotransplantation, where this epitope is expressed on the surface of pig cells and causes HAR (Sandrin et al., 1993
). Hence the expression of the targeted galdase in a transgenic pig could reduce cell surface expression of Gal
(1,3)Gal. We have shown here that galdase-furin(Tm+Cyto) can be effective at decreasing cell surface Gal
(1,3)Gal on PIECs (Figure 7) and also that at least at the expression levels observed, it is more effective at decreasing cell surface Gal
(1,3)Gal in a whole transgenic animal. Unfortunately, at the expression levels demonstrated in this article, the galdase-furin(Tm+Cyto), expressed by itself, would probably be insufficient in a transgenic pig at eliminating HAR. However, other refinements of the strategy could address this problem. One would be increasing the expression level of the transgene. A recently generated galdase-trangenic mouse line (Kase et al., 1998
) with extremely high expression levels of galdase, over 10-fold higher than achieved here, has no lethal effects or abnormalities in the mouse, indicating that the potential exists for increased expression levels of the enzyme. Another possibility is the coexpression of the transgene with other Gal
(1,3)Gal-modifying enzymes. The results (Figure 8D), with galdase-furin(Cyto-Tm) expressed in conjunction with
(1,2)FT, show merit in the effectiveness of this approach because the combination had an additive effect in reducing Gal
(1,3)Gal compared with either transgene expressed in hemizygote mice.
Other factors could dictate the success or failure of using galdase chimeras, such as the microenvironment of the enzyme, particularly pH. The activity of galdase is greatest at pH 4.5 (Desnick et al., 1973) which is appropriate to its function as a glycosidase within lysosomes but not as a glycosidase in the TGN, which is less acidic (pH 6.0) or at the plasma membrane (pH 7.0). Thus, an important factor for further consideration is the suitability of the final destination to its enzyme activity. It remains to be determined whether mutations of the galdase catalytic domain, which can alter its activity at different pH levels (Ishii et al., 1995
) or targeting galdase derived from a species other than humans that have greater activity at different pH will prove to be a more useful for the elimination of Gal
(1,3)Gal in combination with our targeting strategy.
Perhaps more important, the targeting of galdase could be useful in enzyme replacement therapy for patients with Fabrys disease, an X-linked inherited disorder of glycosphingolipid metabolism that leads to the accumulation of ceramide trihexoside and other glycolipids in tissues (Brady et al., 1967). Enzyme replacement therapy has been used in the treatment of this disease (Medin et al., 1996
; Schiffmann et al., 2000
); however, the enzyme has a short half-life and is rapidly cleared from the circulation. Gene replacement therapy has also been undertaken in vitro using retroviral vectors with some limited success (Medin et al., 1996
). It could be useful to employ gene replacement therapy based on a chimeric galdase containing endosomal targeting signals and retrieval signals of furin or other targeting signals attached to a pH-modified galdase. This could allow the degalactosylation of accumulated glycolipids by an active chimeric galdase such that the degradation of these glycolipids can resume, and the amount of intracellular deposits could be reduced.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Generation of galdase constructs
The constructs generated are shown (Figure 1) and were made using standard DNA procedures (Ausubel et al., 1987; Sambrook and Maniatis, 1989
). The human galdase cDNA in the vector pAsc.8 was a generous gift from R. J. Desnick and was subcloned into pcDNA1 (Invitrogen, Groningen, The Netherlands) to produce the plasmid p-galdase (Figure 1) as described previously (Osman et al., 1997
). Furin sequences were generated as follows: a 597-bp fragment from exon 8 of human furin (Roebroek et al., 1986
) was amplified by polymerase chain reaction (PCR) from human genomic DNA using oligonucleotide primer pair MO123 5'TATAGCACCGAGAATGACGTG3' (nt 27372757) and MO124 5'CCAAACCCAGTCCCAAGATAA3' (nt 33133333). A second-step PCR amplified an internal 260-bp fragment consisting of the furin transmembrane and cytoplasmic coding sequence including the stop codon using the primer pair GTM7 5'CATGAATTCGAGGTGGTGGCCGGCCTCA3'(nt 30013019 plus EcoRI site underscored) and GTM4 5'CATCTGCAGTCAGAGGGCGCTCTGGTCTTT3' (nt 32173243 plus a PstI site underscored and stop codon in italics), the fragment furin(Tm+Cyto). A second internal 211-bp fragment containing the furin transmembrane and 5' flanking region was amplified using the primer pair GTM3 (described already) and GTM8 5'CGTCTAGATCATCAGCGCAGCTGCAGGACC3' (nt 30603075, XbaI site underscored and stop codon in italics), the fragment furin(Tm). Both fragments, furin(Tm+Cyto) and furin(Tm), were subcloned by TA cloning (Holton and Graham, 1991
) into the pMosblue vector (Amersham). The chimeric constructs were made as follows. The CD7 stalk sequence (147 bp) was generated by PCR from CD7 in CDM8 (Aruffo and Seed, 1987
) using the primer pair GTM5 5'CATGGATCCCAAGGATGGCACAGATGC3' (nt 895915 plus BamHI site underscored) and GTM6 5'CATGAATTCTCCCTCCCGTCTCCGAC3' (nt 9851004 plus EcoRI site underscored). The PCR product was digested with BamHI/EcoRI, purified, and then ligated into BamHI/EcoRI-digested p-galdase to produce the intermediate p-galdase-CD7. p-Galdase-furin(Tm+Cyto) (Figure 1), was generated by ligating the EcoRI/XbaI-digested 260-bp furin(Tm+Cyto) fragment into EcoRI/XbaI-digested p-galdase-CD7. p-Galdase-furin(Tm) (Figure 1) was produced by ligating purified BamHI/XbaI-digested 211-bp furin(Tm) fragment into BamHI/XbaI-digested p-galdase. The sequences of all chimeric constructs were confirmed by sequencing both strands using the ABI automated sequencer 377 (Applied Biosystems). To produce a stable expressing cell line of galdase-furin(Tm+Cyto) and galdase, PIEC was used. The galdase-furin(Tm+Cyto) fragment was subcloned into pCDNA1(Neo) (Invitrogen) by the HindIII/XbaI sites, and for galdase, the pAsc.8 vector containing the galdase cDNA was used.
Transfection and immunofluorescence
COS-7 cells were maintained in Dulbeccos modified Eagles medium (DMEM) (Cytosystems) and were transfected using the DEAE-dextran transfection method as described elsewhere (Osman et al., 1997). PIECs were maintained in DMEM and were transfected using CaPO4 transfection as described (Wigler et al., 1979
). The transfected cells were selected by growing in DMEM containing G418 at 2 mg/ml for 3 weeks, and a number of clones were selected based on their reduction in cell surface Gal
(1,3)Gal and galdase enzyme activity. CHOP cells (Heffernan and Dennis, 1991
) were maintained in DMEM and transfected with Lipofectamine Plus regent (Gibco BRL Life Technologies). Briefly, 3 x 105 cells were cultured overnight, subsequently transfected using a total of 2.5 µg of plasmid DNA for 2.5 h, washed, then cultured for a further 48 h. Transfection efficiencies of each plasmid were determined by immunofluresence staining for the relevant expressed protein and counting the number of cells that were positive. All constructs were found to have equivalent transfection efficiencies at the plasmid concentration used for transfections. The cells were examined for Gal
(1,3)Gal expression by cytofluorographic analysis on a FACSCalibur flow cytometer (Becton Dickinson).
For confocal microscopy analysis, transfected cells were grown in four-well plastic Lab Tek II chamber slides (Nalge Nunc). A two-stage formaldehyde fixation method (Pawley, 1995) involved fixing the cells in a 2% paraformaldehyde (BDH Laboratory Supplies) solution, first at pH 6.5 and then at pH 10. In some experiments cells were treated in culture before fixation with cycloheximide and BFA (Sigma). For cycloheximide (Sigma) treatment, cells were incubated with cycloheximide (150 µg/ml) for 2 h; for BFA treatment cells were first treated with cycloheximide and then BFA (10 µg/ml) for 10 min. Fixed cells were washed with phosphate-buffered saline (PBS), pH 7.2, containing 0.5% bovine serum albumin (BSA) and permeabilized with 0.1% v/v Triton X-100 (Merck) in PBS. For indirect immunofluorescence, the cells were incubated with primary antibodies (such as anti-
-adaptin, anti-galdase, in PBS/0.5% BSA for 30 min on ice), washed twice, incubated with secondary antibody for 30 min on ice, washed, air-dried, mounted in Prolong Antifade (Molecular Probes), and visualized using an Optiscan krypton/argon laser confocal microscope (Optiscan). Excitation wavelengths of 488 nm and 568 nm were used for FITC and X-Texas red fluorescence, respectively.
Galdase assays
Lysates from transfected cells were assayed for galdase activity using 4-methylumbelliferyl--D-galactopyranoside (Bishop and Desnick, 1981
), and protein concentration was estimated using a Bradford assay. The results shown are from at least three experiments.
Production of the transgenic construct, purification, and microinjection
A 1697-bp NruI/NotI DNA fragment encoding the full-length galdase-furin(Tm+Cyto) was generated utilizing PCR and the galdase-furin(Tm+Cyto) plasmid. The 5' primer homologous to the 5' untranslated region was 5'-TCGCGAatgcagctgaggaacccagaa, in which the underscored sequence contains a unique NruI site, and the 3' primer homologous to the 3' untranslated region was 5'-CcaggacgtcgacgcgactactCGCCGGCGtag (the underscored sequence contains a NotI site). The DNA was separated by electrophoresis in agarose gels, purified using QIAEX II beads (Qiagen), and subcloned into a NruI/NotI digested genomic H-2Kb-containing vector (12), resulting in the plasmid clone (pH-2Kb-galdase-furin(Tm+Cyto)) encoding the galdase-furin(Tm+Cyto) gene directionally cloned into exon 1 of the murine H-2Kb gene. The construct was engineered such that translation would begin at the initiation codon (ATG) of the galdase-furin(Tm+Cyto) cDNA and terminate at the stop codon (TGA) 1686 bp downstream. DNA was prepared for microinjection by digesting pH-2Kb-galdase-furin(Tm+Cyto) with XhoI and purification of the H-2Kb-galdase-furin(Tm+Cyto) DNA from vector by electrophoretic separation in agarose gels, and purified using QIAEX II beads. Injections were performed into the pronuclear membrane of (C57BL/6xSJL)F1 zygotes at concentrations between 2 and 5 ng/µl, and the zygotes were transferred to pseudopregnant (C57BL/6xSJL)F1 females as previously described (Cohney et al., 1997).
Screening for the transgene
The presence of the transgene in the live offspring was sought by a combination of Southern blotting and dot blotting. (1) For Southern blotting, genomic DNA (~10 µg) was isolated from the tail and digested to completion with BamHI; the DNA was separated by electrophoresis on a 0.8% agarose gel, transferred to nylon filters, Hybond N+ (Amersham), and hybridized with the furin(Tm+Cyto) portion from p-galdase-furin(Tm+Cyto), using a final wash at 68°C in 0.1x 3M sodium chloride 0.3M trisodium citrate pH 7.0/1% sodium dodecyl sulfate. (2) For dot blots, 2 µg and 5 µg of genomic DNA was transferred to nylon filters Hybond (Amersham), hybridized, and washed as described. Homozygous and hemizygous transgenic mice were distinguished by comparing the density of galdase probed autoradiographs obtained from Southern blots and standardizing the probing with the murine (1,3)GT gene (Larsen et al., 1989
) to determine relative copy number.
![]() |
Acknowledgments |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ausubel, F.M.B., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (1987) Current protocols in molecular biology. Wiley, New York.
Bishop, D.F. and Desnick, R.J. (1981) Affinity purification of alpha-galactosidase A from human spleen, placenta, and plasma with elimination of pyrogen contamination. Properties of the purified splenic enzyme compared to other forms. J. Biol. Chem., 256, 13071316.
Bosshart, H., Humphrey, J., Deignan, E., Davidson, J., Drazba, J., Yuan, L., Oorschot, V., Peters, P.I., and Bonifacino, J.S. (1994) The cytoplasmic domain mediates localization of furin to the trans-Golgi network en route to the endosomal/lysosomal system. J. Cell Biol., 126, 11571172.[Abstract]
Brady, R.O., Gal, A.E., Bradley, R.M., and Martensson, E. (1967) The metabolism of ceramide trihexosides. I. Purification and properties of an enzyme that cleaves the terminal galactose molecule of galactosylgalactosylglucosylceramide. J. Biol. Chem., 242, 10211026.
Cohney, S., McKenzie, I.F., Patton, K., Prenzoska, J., Ostenreid, K., Fodor, W.L., and Sandrin, M.S. (1997) Down-regulation of Gal alpha(1, 3)Gal expression by alpha1, 2-fucosyltransferase: further characterization of alpha1, 2-fucosyltransferase transgenic mice. Transplantation, 64, 495500.[ISI][Medline]
Desnick, R.J., Allen, K.Y., Desnick, S.J., Raman, M.K., Bernlohr, R.W., and Krivit, W. (1973) Fabrys disease: enzymatic diagnosis of hemizygotes and heterozygotes. Alpha-galactosidase activities in plasma, serum, urine, and leukocytes. J. Lab. Clin. Med., 81, 157171.[ISI][Medline]
Heffernan, M. and Dennis, J.W. (1991) Polyoma and hamster papovavirus large T antigen-mediated replication of expression shuttle vectors in Chinese hamster ovary cells. Nucleic Acids Res., 19, 8592.[Abstract]
Holton, T.A. and Graham, M.W. (1991) A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors. Nucleic Acids Res., 19, 1156.[ISI][Medline]
Ioannou, Y.A., Bishop, D.F., and Desnick, R.J. (1992) Overexpression of human alpha-galactosidase A results in its intracellular aggregation, crystallization in lysosomes, and selective secretion. J. Cell Biol., 119, 11371150.[Abstract]
Ioannou, Y.A., Zeidner, K.M., Grace, M.E., and Desnick, R.J. (1998) Human alpha-galactosidase A: glycosylation site 3 is essential for enzyme solubility. Biochem. J., 332, 789797.[ISI][Medline]
Ishii, S., Kase, R., Sakuraba, H., and Suzuki, Y. (1995) The functional role of glutamine-280 and threonine-282 in human alpha-galactosidase. Biochim. Biophys. Acta, 1270, 163167.[ISI][Medline]
Jones, B.G., Thomas, L., Molloy, S.S., Thulin, C.D., Fry, M.D., Walsh, K.A., and Thomas, G. (1995) Intracellular trafficking of furin is modulated by the phosphorylation state of a casein kinase II site in its cytoplasmic tail. EMBO J., 14, 58695883.[Abstract]
Kase, R., Shimmoto, M., Itoh, K., Utsumi, K., Kotani, M., Taya, C., Yonekawa, H., and Sakuraba, H. (1998) Immunohistochemical characterization of transgenic mice highly expressing human lysosomal alpha-galactosidase. Biochim. Biophys. Acta, 1406, 260266.[ISI][Medline]
Larsen, R.D., Rajan, V.P., Ruff, M.M., Kukowska-Latallo, J., Cummings, R.D., and Lowe, J.B. (1989) Isolation of a cDNA encoding a murine UDPgalactose:beta-D-galactosyl-1, 4-N-acetyl-D-glucosaminide alpha-1, 3-galactosyltransferase: expression cloning by gene transfer. Proc. Natl Acad. Sci. USA, 86, 82278231.[Abstract]
Medin, J.A., Tudor, M., Simovich, R., Quirk, J.M., Jacobsen, S., Murray, G.J., and Brady, R.O. (1996) Correction in trans for Fabry disease: expression, secretion and uptake of alpha-galactosidase A in patient-derived cells driven by a high- titer recombinant retroviral vector. Proc. Natl Acad. Sci. USA, 93, 79177922.
Molloy, S.S., Thomas, L., VanSlyke, J.K., Stenberg, P.E., and Thomas, G. (1994) Intracellular trafficking and activation of the furin proprotein convertase: localization to the TGN and recycling from the cell surface. EMBO J., 13, 1833.[Abstract]
Osman, N., McKenzie, I.F., Ostenreid, K., Ioannou, Y.A., Desnick, R.J., and Sandrin, M.S. (1997) Combined transgenic expression of alpha-galactosidase and alpha1, 2- fucosyltransferase leads to optimal reduction in the major xenoepitope Galalpha(1, 3)Gal. Proc. Natl Acad. Sci. USA, 94, 1467714682.
Pawley, J.B. (1995) Handbook of biological confocal microscopy. Plenum, New York.
Reaves, B. and Banting, G. (1992) Perturbation of the morphology of the trans-Golgi network following brefeldin A treatment: redistribution of a TGN-specific integral membrane protein, TGN38. J. Cell Biol., 116, 8594.[Abstract]
Roebroek, A.J., Schalken, J.A., Leunissen, J.A., Onnekink, C., Bloemers, H.P., and Van den Ven, W.J. (1986) Evolutionary conserved close linkage of the c-fes/fps proto-oncogene and genetic sequences encoding a receptor-like protein. EMBO J., 5, 21972202.[Abstract]
Sambrook, J.F. and Maniatis, E.F. (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Sandrin, M.S. and McKenzie, I.F. (1999) Recent advances in xenotransplantation. Curr. Opin. Immunol., 11, 527531.[CrossRef][ISI][Medline]
Sandrin, M.S., Fodor, W.L., Mouhtouris, E., Osman, N., Cohney, S., Rollins, S.A., Guilmette, E.R., Setter, E., Squinto, S.P., and McKenzie, I.F. (1995) Enzymatic remodelling of the carbohydrate surface of a xenogenic cell substantially reduces human antibody binding and complement-mediated cytolysis. Nat. Med., 1, 12611267.[ISI][Medline]
Sandrin, M.S., Vaughan, H.A., Dabkowski, P.L., and McKenzie, I.F. (1993) Anti-pig IgM antibodies in human serum react predominantly with Gal(alpha 1-3)Gal epitopes. Proc. Natl Acad. Sci. USA, 90, 1139111395.[Abstract]
Schafer, W., Stroh, A., Berghofer, S., Seiler, J., Vey, M., Kruse, M.L., Kern, H.F., Klenk, H.D., and Garten, W. (1995) Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin. EMBO J., 14, 24242435.[Abstract]
Schiffmann, R., Murray, G.J., Treco, D., Daniel, P., Sellos-Moura, M., Myers, M., Quirk, J.M., Zirzow, G.C., Borowski, M., Loveday, K., and others. (2000) Infusion of alpha-galactosidase A reduces tissue globotriaosylceramide storage in patients with Fabry disease. Proc. Natl Acad. Sci. USA, 97, 365370.
Shapiro, J., Sciaky, N., Lee, J., Bosshart, H., Angeletti, R.H., and Bonifacino, J.S. (1997) Localization of endogenous furin in cultured cell lines. J. Histochem. Cytochem., 45, 312.
Stanley, P., Sudo, T., and Carver, J.P. (1980) Differential involvement of cell surface sialic acid residues in wheat germ agglutinin binding to parental and wheat germ agglutinin-resistant Chinese hamster ovary cells. J. Cell Biol., 85, 6069.[Abstract]
Teuchert, M., Schafer, W., Berghofer, S., Hoflack, B., Klenk, H.D., and Garten, W. (1999) Sorting of furin at the trans-Golgi network. Interaction of the cytoplasmic tail sorting signals with AP-1 Golgi-specific assembly proteins. J. Biol. Chem., 274, 81998207.
Vaughan, H.A., Henning, M.M., Purcell, D.F., McKenzie, I.F., and Sandrin, M.S. (1991) The isolation of cDNA clones for CD48. Immunogenetics, 33, 113117[ISI][Medline]
Wigler, M., Sweet, R., Sim, G.K., Wold, B., Pellicer, A., Lacy, E., Maniatis, T., Silverstein, S., and Axel, R. (1979) Transformation of mammalian cells with genes from procaryotes and eucaryotes. Cell, 16, 777785.[ISI][Medline]