Laminin receptor (LR) is a 67-kd cell surface protein that binds laminin with high affinity in mammals (Malinoff and Wicha, 1983; Rao et al., 1983). LR is expressed in a wide variety of cells, especially abundant in cancers (Hand et al., 1985; Yow et al., 1988; Sobel, 1993; Sanjuan et al., 1996; Menard et al., 1997). This has led to the suggestion that LR is involved in tumor development and cancer progression.
Although LR has no obvious signal peptide and the mammalian 37-kd precursor of LR remains in the cytoplasm as a component of the translational machinery (Auth and Brawerman, 1992; Ardini et al., 1998), cell surface association of mature LR has been well documented (Castronovo et al., 1991; Starkey et al., 1999). It is unclear how the 37-kd precursor is converted to the 67-kd LR, although it has been suggested that it is due to either homo- or heterodimerization of the 37-kd LR (Landowski et al., 1995; Menard et al., 1997; Buto et al., 1998) (for simplicity, we will refer to both the precursor and mature protein as LR in this report). Regardless, in the membrane bound state, the N-terminal third localizes to the cytoplasm while the C-terminal two-thirds of the 37-kd LR is located extracellularly and contains a 6 amino acid laminin-binding sequence (Castronovo et al., 1991), thus enabling cell surface LR to bind to laminin in the extracellular matrix (ECM).
We have been studying the role of matrix metalloproteinases (MMPs) during vertebrate development by using thyroid hormone-dependent intestinal remodeling during amphibian metamorphosis as a model. MMPs are a large family of zinc-dependent, extracellular, or membrane-bound proteases (Alexander and Werb, 1991; Birkedal-Hansen et al., 1993; Barrett et al., 1998; Nagase, 1998; Parks and Mecham, 1998; Pei, 1999; McCawley and Matrisian, 2001). Collectively, they are capable of cleaving all protein components of the ECM. In addition, MMPs are also capable of degrading non-ECM extracellular or membrane-bound proteins. On the other hand, in the vast majority of the cases, it is unknown whether MMPs cleave these in vitro substrates during normal or pathological processes in vivo.
Intestinal remodeling during amphibian metamorphosis offers a unique opportunity to study the role of MMPs in postembryonic development (Shi and Ishizuya-Oka, 2001). Tadpole intestine is a simple tubular organ with a single layer of larval epithelium surrounded by thin layers of connective tissue and muscles (Shi and Ishizuya-Oka, 1996). In the course of metamorphosis, massive apoptosis of the larval epithelium and concurrent proliferation and differentiation of the adult epithelium are accompanied by the remodeling of basement membrane or basal lamina, the ECM that separates the epithelium from the connective tissue. These changes lead to the formation of a multiply-folded frog epithelium surrounded by elaborate connective tissue and thick muscle layers. Like all other transformations during metamorphosis, intestinal remodeling is controlled by thyroid hormone (T3). Blocking the synthesis of endogenous T3 prevents its metamorphosis while addition of exogenous T3 to premetamorphic tadpole rearing water or even organ culture medium leads to precocious transformations, making it an excellent model to isolate and functionally characterize genes important for postembryonic organogenesis.
By isolating genes that are regulated by T3, we and others have previously shown that a number of MMPs are upregulated during metamorphosis (Shi, 1999; Amano et al., 2003). In particular, the expression of ST3 is correlated with cell death in different organs during metamorphosis in Xenopus laevis (Patterton et al., 1995; Ishizuya-Oka et al., 1996; Berry et al., 1998a, b; Damjanovski et al., 1999; Wei and Shi, 2005). More importantly, in the animal intestine, we have shown that ST3 expression precedes the remodeling of the basal lamina, apoptosis in the larval epithelium, and morphogenesis of the adult intestinal epithelium. Using intestinal organ cultures in conjunction with a function-blocking anti-ST3 antibody added into the culture medium, we have shown that ST3 function is required for not only basal lamina remodeling, but also larval epithelial cell death and adult cell migration (Ishizuya-Oka et al., 2000).
To investigate how ST3 affects intestinal remodeling, we have recently isolated Xenopus laevis LR as an in vitro substrate of ST3 by using a yeast-two-hybrid screen. Here we report that LR is expressed during intestinal remodeling but is regulated in a tissue- and developmental-stage dependent manner. Furthermore, during metamorphosis when ST3 is highly expressed, LR is cleaved to fragments of sizes expected from ST3 cleavage. More importantly, transgenic expression of ST3 in premetamorphic tadpoles also leads to precocious LR cleavage in the intestine. Taken together, we conclude that LR plays a role during intestinal remodeling through tissue-dependent expression and cleavage by ST3.
LR Is Expressed in Different Organs and Throughout Intestinal Development
To investigate the potential role of LR during frog development, we examined LR expression in Xenopus tadpoles. The polyclonal anti-Xenopus LR antibody detected a single band migrating at 47 kd in all organs/tissues of premetamorphic tadpoles at stage 56/57 (Fig. 1). (Note that the gene is named 37-kd laminin receptor precursor even though its apparent size on the gel is larger.) This antibody failed to detect the 67-kd form of LR on Western blot, which might not be surprising as mammalian 67-kd and the 37-kd precursor also have distinct antigenicity (Rao et al., 1989; Castronovo et al., 1991).
To study the developmental expression profiles, we focused on the intestine because of its interesting remodeling process as described above. First, Northern blot analysis showed that LR mRNA was expressed at fairly constant levels throughout development (Fig. 2A). For comparison, we also analyzed the expression of the matrix metalloproteinase ST3, which, in contrast, was at high levels exclusively at metamorphic climax (stages 60–62) (Fig. 2A). Similarly, Western blot analysis revealed the expression of full-length LR protein throughout intestinal development (Fig. 2B). Interestingly, the anti-Xenopus LR antibody recognized a few minor, smaller bands in addition to the major 47-kd full-length band during development (Fig. 2B). While the largest of them (Fig. 2B, triangle) was observed at various stages in some replicate experiments, the two smaller bands were observed only at the climax of intestinal remodeling (stage 61, Fig. 2B, lane 4, filled or open circle). These bands were of similar sizes as the C-terminal fragments of LR generated from in vitro ST3 cleavage (Amano et al., 2005) and their appearance coincided with high levels of ST3 expression during development (Patterton et al., 1995), suggesting that LR may be cleaved in vivo during intestinal metamorphosis by ST3 (also see below).
LR Expression Is Regulated in a Tissue-Specific Manner During Intestinal Metamorphosis
The intestine is made of multiple tissues although the epithelium is the predominant one in tadpoles. Thus, while overall expression of LR did not change during intestinal metamorphosis, its spatial expression pattern may change. To analyze this, immunohistochemical studies were carried out on sections of the small intestine from tadpoles at different stages. Prior to the onset of metamorphic climax (stage 57/58), LR was expressed only in the larval epithelium (Fig. 3, st 57). By stage 60, when the larval epithelial cells undergo apoptosis and adult epithelial cells as well as fibroblasts and muscle cells proliferate (Shi and Ishizuya-Oka, 1996), some fibroblasts became positive in addition to the strong expression in the larval epithelium. At this stage, precursors for adult epithelium appeared as a small cluster of proliferating cells and these adult primordia did not express or only very weakly expressed LR (Fig. 3, st 60). During the progression of intestinal remodeling, LR-negative proliferating adult primordia gradually replaced the LR-positive larval epithelium, while the expression in the connective tissue became strong (Fig. 3, st 62). As the adult epithelial cells differentiated and intestinal folds formed by stage 64 (Fig. 3, st 64), some adult epithelial cells became LR-positive and fibroblasts remained LR positive. After completion of metamorphosis, no expression of LR in fibroblasts was detected, and LR expression became limited to the differentiated epithelium (Fig. 3, st 66). Throughout intestinal remodeling, the muscle layers had little or weak expression of LR.
Intestinal remodeling, like other processes during metamorphosis, can be induced by treating premetamorphic tadpoles with exogenous T3. If the spatiotemporal changes in LR expression play a role in intestinal remodeling, we would expect that they should be induced by T3 treatment of premetamorphic tadpoles. Thus, we treated premetamorphic tadpoles at stage 55/56 with 5 nM of T3. Again, in the untreated animal intestine, LR was expressed in the larval epithelium but not in the fibroblasts of the connective tissue (Fig. 4). After 5 days of treatment, the fibroblasts strongly expressed LR (Fig. 4). After 7 days, adult epithelium replaced most of the larval epithelium and had little or no staining and fibroblasts continued to express LR as during natural metamorphosis.
ST3 Cleaves Cell Surface LR
The Xenopus LR was first isolated as a putative substrate of MMP ST3 (Amano et al., 2005). In vitro studies have clearly shown that ST3 cleaves LR at two sites in the extracellular domain between the laminin binding sequence and the transmembrane domain (Fig. 5A) (Amano et al., 2005). If LR is an in vivo substrate of ST3 during metamorphosis, ST3 would be expected to cleave membrane-bound LR in either the tadpole epithelial cells or the proliferating fibroblasts during intestinal remodeling. To determine whether ST3 cleaves membrane bound LR, we overexpressed C-terminally Flag-tagged Xenopus LR in Cos7 cells by transfection. Three days after transfection, the culture medium was changed to a low serum medium containing Zn2+, and purified ST3Cw or ST3Cm was added to the culture medium. To concentrate C-terminal degradation products released into culture medium, Flag-tagged proteins were precipitated with anti-Flag antibody beads. Both the whole cell lysate and immunoprecipitated proteins were subjected to Western blot using a polyclonal anti-LR antibody raised against purified Xenopus LR. The expected C-terminal degradation products of LR were seen from the culture medium of Cos7 cells treated with ST3Cw but not ST3Cm (Fig. 5B). In addition, no C-terminal degradation products were detected in the whole cell lysate. These results suggest that ST3 cleaves cell surface LR. It is worth noting, however, that there was a significant amount of full-length LR detected in the immunoprecipitate. The full-length LR was likely due to cell lysis during protein isolation, although some may be due to shedding from cell surface (Karpatova et al., 1996). In the absence of proper reagents or methods to definitively resolve this, it is possible that some of the cleavage products might be due to the cleavage of the shed LR by ST3. Nonetheless, our results here are consistent with the ability of ST3 to cleave cell surface LR.
LR Is Cleaved by ST3 in the Intestine During Development
Our developmental Western blot analysis above suggests that LR is cleaved during intestinal remodeling (Fig. 2B). The sizes of the LR fragments and their presence predominantly during the climax of intestinal remodeling when ST3 is expressed suggest that LR is cleaved by ST3 during development. To confirm this in vivo cleavage of LR by ST3, we generated transgenic tadpoles expressing a fusion protein of full-length ST3 with the green fluorescent protein (GFP) at the C-terminus under the control of a heat shock inducible promoter (Fu et al., 2002). After heat shock treatment for 2–3 days of wild type or transgenic tadpoles at stage 55/56 in the same tank, transgenic tadpoles were identified based on GFP fluorescence. Transgenic animals had green eyes and body under fluorescent microscope due to GFP expression in the eye driven by the γ-crystalline promoter and ST3-GFP expression in the whole body driven by the heat shock promoter (Fu et al., 2002). [Unlike embryonic overexpression of ST3, which leads to severe developmental defects and lethality (Fu et al., 2002), overexpression of ST3 under the heat shock inducible promoter at the premetamorphic tadpole stages does not produce overt morphological changes, although after 4–7 days, some intestinal remodeling events are induced (Fu et al., 2005).] Total proteins were isolated from the small intestine of each group of tadpoles and subjected to Western blot analysis. For comparison, we also analyzed intestinal proteins from premetamorphic tadpoles at stage 57 and metamorphosing tadpoles at stage 61. As with wild type animals at metamorphic climax (stage 61), we found two bands of similar sizes as those generated from in vitro cleavage of LR by ST3 in transgenic but not wild type animals (Fig. 6A, circles) (the two higher molecular weight bands under the full-length LR were also present in wild type animals at different stages, although their levels were higher in transgenic animals). Furthermore, more LR cleavage was observed in animals treated with heat shock for 3 days than those for 2 days as expected (Fig. 6A, compare lane 6 to 4).
Interestingly, of the two likely ST3 cleavage products, the lower band (open circle) was stronger in transgenic animals than the upper band, especially after 3 days of heat shock treatment (solid circle, Fig. 6A, lanes 4 and 6), opposite to that observed at the climax of metamorphosis (Fig. 6A, lane 2). This is likely due to higher levels of ST3 in transgenic animals compared to that at the climax of metamorphosis, thereby leading to more complete digestion of LR to produce the shorter form (double digestion results in a shorter form while single digestion produces either one, see Fig. 5A).
To obtain further evidence that the in vivo LR fragments were from ST3 cleavage, we analyzed side by side the protein extracts from wild type animals at the climax of metamorphosis, transgenic animals, tissue culture cells transfected with LR expression plasmid and treated with ST3, and LR cleaved by ST3 in vitro. As expected, the full-length LR from tadpoles was smaller than the in vitro translated LR or LR from Cos7 cells due to the presence of N-terminal tag (52a.a.) in the in vitro translated LR and both N- and C-terminal tags (34a.a. and 8a.a., respectively) in the LR from Cos7 cells (Fig. 6B). The C-terminal cleavage products from Cos7 cells were slightly larger than the in vitro products due to the presence of the C-terminal tag and possible posttranslational modifications. The C-terminal cleavage products of LR from the intestine of metamorphosing tadpoles (stage 61, Fig. 6B, lane 1) or transgenic tadpoles expressing ST3 (Fig. 6B, lane 2) were of similar sizes to those from Cos7 cells (Fig. 6B, open and solid circles). Considering posttranslational modifications and the fact that cleavage of LR by other MMPs that we have tested (Amano et al., 2005) produce LR products of very different sizes than what we observed in vivo, the results here strongly argue that the LR fragments in metamorphosing animals or transgenic tadpoles were due to in vivo cleavage of LR by ST3 at the same sites as observed in vitro (Amano et al., 2005).
LR was first identified as a 67-kd protein that binds laminin with high affinity (Malinoff and Wicha, 1983; Rao et al., 1983). The LR gene encodes a protein of 37 kd. Most of the 37-kd LR stays in the cytoplasm as a component of the translational machinery (Auth and Brawerman, 1992; Ardini et al., 1998) and a fraction is converted into the 67-kd, cell surface–associated mature LR through either homo- or heterodimerization of the 37-kd LR (Castronovo et al., 1991; Landowski et al., 1995; Menard et al., 1997; Buto et al., 1998; Starkey et al., 1999). LR is expressed in a wide variety of cells, particularly in cancers (Menard et al., 1997). Little information is, however, known about its role in cell function. The Xenopus LR was recently isolated as a putative substrate for the matrix metalloproteinase ST3. Our studies here provide evidence to suggest a role of LR in cell fate determination and tissue morphogenesis during development through tissue-dependent developmental regulation of its expression and through its cleavage by ST3.
Cell Surface LR Is an In Vivo Substrate of ST3
The matrix metalloproteinase stromelysin-3 was first isolated from a human breast tumor in which ST3 was highly expressed in the stromal cells surrounding neoplastic cells and has since been shown to be expressed in a number of developmental and pathological processes (Basset et al., 1990, 1997; Lefebvre et al., 1992, 1995; Ahmad et al., 1998; Damjanovski et al., 1999; Lochter and Bissell, 1999; Shi and Ishizuya-Oka, 2001; Wei and Shi, 2005). Such extensive studies on ST3 expression have led to the suggestion that ST3 regulates cell fate and behavior although little is known as to how ST3 may function in this manner.
Extensive biochemical studies have shown that in vitro, MMPs can cleave various proteinaceous components of the ECM. In addition, more recent studies have shown that in vitro, MMPs are also capable of cleaving non-ECM extracellular proteins (Barrett et al., 1998; Uria and Werb, 1998; McCawley and Matrisian, 2001; Overall, 2002). However, it has been much more challenging to study substrate cleavage by MMPs in vivo, largely due to the difficulty of detecting the cleaved products. A combination of molecular, genetic, and cell biological studies have provided strong evidence to support the cleavage of type I collagen by collagenase 1 (MMP1) or other collagenases in vivo (Wu et al., 1990; Liu et al., 1995; Pilcher et al., 1997). In addition, by using mouse deficient in matrilysin (MMP7), the intestinal paneth cell α-defensins (cryptdins) and epithelial cell surface-bound proteoglycan syndecan-1 were identified as in vivo substrates of this MMP (Wilson et al., 1999; Li et al., 2002), aided in part by the formation of stable cleavage products.
Unlike essentially all other MMPs, ST3 does not degrade any ECM proteins efficiently but cleaves the non-ECM proteins α1PI and IGFBP-1 in vitro (Murphy et al., 1993; Pei et al., 1994; Manes et al., 1997; Overall, 2002). Our recent isolation of LR as an in vitro substrate of ST3 raises a possibility that ST3 may function in part by cleaving cell surface LR, thereby altering cell-ECM (extracellular matrix interaction) (Amano et al., 2005). Our studies here have provided several lines of evidence to support such a mechanism.
First, we used amphibian metamorphosis as a model to obtain evidence that ST3 cleaves LR during development. Amphibian metamorphosis transforms essentially every organ and tissue of the animal during a short developmental period (Shi, 1999). Of particular interest is the transformation of the animal intestine. The simple tubular organ of predominantly a single layer of larval/tadpole epithelial cells in premetamorphic tadpoles is transformed within several days to a complex one of a multiply folded epithelium surrounded by elaborated connective tissue and muscles (Shi and Ishizuya-Oka, 1996). This complete change of the entire organ within a short period thus offers an opportunity to detect potential cleavage intermediates of any substrates by ST3. Indeed, by analyzing the developmental expression profiles of LR in the intestine during metamorphosis, we obtained the first evidence to suggest that LR is cleaved by ST3 during intestinal remodeling. That is, the presence of two fragments of LR of similar lengths to the C-terminal fragments of LR cleaved by ST3 in vitro coincides with the high levels of ST3 expression during metamorphosis.
Second, direct evidence to support LR cleavage by ST3 in vivo came from our transgenic studies where precocious overexpression of ST3 alone through transgenesis in premetamorphic tadpoles was able to produce the same two LR fragments. Finally, when Cos7 cells were transfected with an LR overexpression plasmid and incubated with purified ST3, we detected the formation of the LR cleavage fragments by ST3 in the cultured medium but not cell lysate, suggesting that ST3 cleaved cell surface–bound LR. The fragments from Cos7 cells or tadpole intestine were of slightly different lengths from each other and from those derived from in vitro cleavage of E. coli produced LR by ST3. This is likely due to different posttranslational modifications of LR in tadpoles and in Cos7 cells as it is well known that LR is modified in vivo and that one modification, i.e., acylation, is known to be important to form mature LR on cell surface (Landowski et al., 1995; Menard et al., 1997; Buto et al., 1998). The confirmation of such a possibility would require directly sequencing purified fragments from in vivo, which is impossible at the present time. Regardless, the similar mobility of the fragments together with the increased formation of such fragments in transgenic tadpoles overexpressing ST3 argues that they were derived from in vivo cleavage of LR by ST3. This is further supported by the fact that all other MMPs that we have tested cleave LR at distinct sites that yield fragments of very different sizes than what we observed in vivo (Amano et al., 2005).
A Likely Role of LR Cleavage by ST3 During Intestinal Remodeling
The first evidence for a potential role of LR during frog development came from its interesting expression profiles. LR is expressed in different organs in premetamorphic tadpoles. While its overall expression level does not change significantly, its expression in different tissues of the intestine changes dramatically during intestinal metamorphosis. It is highly expressed in the larval epithelium but not in the connective tissue prior to metamorphosis. During metamorphosis, the LR expressing larval epithelial cells undergo apoptosis and the proliferating adult epithelium does not express LR. Concurrently, LR is expressed in the proliferating connective tissue cells, which also express high levels of ST3 (Patterton et al., 1995). By the end of metamorphosis, the differentiated adult intestinal epithelial cells express LR while connective tissue does not.
The expression profile of LR during intestinal development together with the high level ST3 expression only during metamorphosis raises the possibility that the differentiated epithelial cells use cell surface LR to interact with laminin in the basal lamina to ensure its survival and/or migration (for adult cells during epithelial morphogenesis toward the end of metamorphosis). The high levels of ST3 expressed during metamorphosis leads to the cleavage of cell surface LR, thereby releasing the cells from the laminin as the cleavage sites are located between the transmembrane domain and laminin binding sequence. This may facilitate larval epithelial cell death. It is worth noting that although only a small fraction of LR was detected as the cleaved form, significant cell surface LR might have been cleaved by ST3. This is because (1) the cleaved fragments might be less stable and thus preferentially degraded over the full length LR and (2) the majority of LR was expected to be in the cytoplasm (Auth and Brawerman, 1992; Ardini et al., 1998). In addition, LR expressed on the surface of the fibroblasts in the connective tissue may also be cleaved by ST3. It is well known that cell-cell contacts between the proliferating adult epithelial cells and fibroblasts occur across the basal lamina and that adult epithelial morphogenesis is likely to involve the migration of both epithelial cells and fibroblasts (Shi and Ishizuya-Oka, 1996). This cleavage of LR on fibroblasts may affect the migration and/or interaction between the fibroblasts adjacent to the basal lamina where laminin is abundant, thus facilitating adult epithelial development. On the other hand, some LR may be shed from the cell surface (Karpatova et al., 1996). The shed LR may also be cleaved by ST3. The extracellular LR fragments produced from cleavage of cell surface LR or shed LR may affect cell fate through other mechanisms as described below. Regardless of the exact mechanisms by which LR cleavage affect intestinal remodeling, such a function appears to be uniquely mediated by ST3. First, ST3 is the only MMP that is known to be dramatically upregulated just prior to and during intestinal remodeling (Patterton et al., 1995; Stolow et al., 1996; Shi and Ishizuya-Oka, 2001; Amano et al., 2003). Secondly, all other MMPs that we have analyzed cleaved LR after the laminin binding sequence; thus, their cleavage of LR, if any, is not expected to affect cell-laminin interaction through LR (Amano et al., 2005).
In addition to its developmental roles, LR is also likely to be involved in tumor development and cancer progression. It is particularly abundant in cancers (Hand et al., 1985; Yow et al., 1988; Sobel, 1993; Sanjuan et al., 1996; Menard et al., 1997). We have shown recently that human LR is also a substrate for ST3 (Amano et al., 2005). Moreover, similar to ST3, there is a strong correlation between LR overexpression and the metastatic property of tumor cells (Castronovo, 1993; Martignone et al., 1993). Thus, the coexistence of ST3, which is expressed in the fibroblasts within the tumors but not actually in tumor cells themselves, and LR in tumors may be expected to lead to the cleavage of tumor cell surface LR. This may alter tumor cell–ECM interaction to affect tumor development and cell migration. In fact, a common function for LR may exist in cancer and intestinal epithelial cells. That is, LR facilitates the survival of both types of cells in vivo. Its cleavage by ST3 may lead to differentiated intestinal epithelial cells to undergo apoptosis while facilitating the proliferative cancer cells to migrate. Interestingly, the lack of LR on the surface of the proliferative adult intestinal epithelial cells, due to the lack of LR expression, may also play a role in their migration during adult epithelial morphogenesis. Thus, the lack or reduced level of cell surface LR may also have a common effect, i.e., promoting migration, on the proliferative cancer and undifferentiated adult intestinal epithelial cells.
How LR affects cell fate and migration remains to be determined. In addition to directly affecting cell–ECM interaction through binding laminin, LR has also been reported to interact with α6β4 integrin and regulate or stabilize the attachment of cells to laminin through integrin (Ardini et al., 1997). The expression level of LR in cancer cells is correlated with abilities to bind laminin (Wewer et al., 1986), which could be due to either the binding of LR directly to laminin or indirectly by affecting integrin-laminin interaction. In addition, LR has been reported to have other functions. Peptide G, which corresponds to the laminin-binding site in LR, increases and stabilizes laminin binding to cancer cells (Magnifico et al., 1996). It can also increase the rate of laminin-1 degradation by the cysteine proteinase cathepsin B to produce a proteolytic fragment that activates cell migration in vitro (Ardini et al., 2002). Therefore it is possible that ST3 cleaves LR to generate physiologically active peptides that contain peptide G sequence to regulate cell fate and behavior. Thus, LR cleavage by ST3 may play a role in the control of cell fate and behavior through different signaling pathways, thereby affecting physiological and pathological processes.
Xenopus laevis tadpoles were staged according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1956). Metamorphosis was induced by adding 5 nM T3 to the rearing water.
ST3 and LR Protein Expression
The cDNA encoding the catalytic domain of wild type (ST3Cw) or enzymatically inactive (ST3Cm) ST3, which was made by introducing a point mutation in the zinc box, resulting in the substitution of amino acid E204 to A204, was subcloned into pET28 (N-terminal His- and T7-tag, Novagen). ST3Cw and ST3Cm were overexpressed in E. coli BL21 (DE3) and purified using Ni-NTA spin columns (Damjanovski et al., 2001).
A cDNA encoding the full-length LR was subcloned into pET30 (N-terminal His- and S-tag, for protein purification) and pCDNA4/HisMax (N-terminal His- and Xpress-tag and additional C-terminal Flag-tag, for Cos7 cell expression, Invitrogen) between Bam H1 and Xho I sites. The protein was overexpressed in E. coli BL21 (DE3) and extracted with 8 M urea, 0.5 M NaCl, 0.1% Triton X-100, 20 mM Tris-Cl, pH 7.9. The extract was incubated with Ni-NTA agarose (Qiagen, Chatsworth, CA) and the protein was eluted with the same buffer containing 200 mM imidazole. Urea and imidazole were removed by dialysis against PBS containing 4, 2, 1, 0.5, and 0 M urea sequentially. The protein solution was finally concentrated by dialysis against 50% glycerol in PBS (Amano et al., 2005).
In Vitro Cleavage of LR
Purified S-tagged LR (2.6 μg) was incubated with 40 ng of ST3Cw in the ST3 reaction buffer (containing 200 mM NaCl, 10 mM CaCl2, 0.1 mM ZnCl2, 0.1% Triton X-100, and 50 mM Tris-Cl, pH 7.4) overnight at room temperature.
ST3 Cleavage of Cell Surface LR Expressed in Cos7 Cells
Cos7 cells were transfected with LR-Flag-pCDNA4/HisMax using FuGENE 6 (Roche) and cultured for 3 days. Cells were incubated with or without ST3Cw or ST3Cm (detergent-free preparation) in DMEM containing 2% FCS, 1 mM CaCl2, and 0.1 mM ZnCl2 at room temperature for 4 hr. Conditioned medium and cells were removed. The conditioned medium was filtered through 0.22-μm filter and incubated with anti-Flag antibody agarose (Sigma, St. Louis, MO) to precipitate any C-terminal peptides of LR resulted from ST3 cleavage of cell surface LR. After several washes with PBS, the proteins were eluted from the beads by SDS-PAGE sample buffer and subjected to Western blotting using anti-Xenopus LR antibody. The cells were lysed in SDS-PAGE sample buffer and similarly analyzed by Western blotting.
Western Blotting, Immunohistochemistry, and Northern Blotting
Anti-Xenopus LR rabbit polyclonal antibodies were raised against purified full-length LR (Covance). Before protein extraction, isolated small intestines were flushed to remove debris in the lumen. Proteins were extracted from various organs at indicated stages of tadpoles with 9 M urea, 5% 2-mercaptoethanol, 1 mM EDTA, and 50 mM Tris-Cl, pH 6.8, containing proteinase inhibitor cocktail (Calbiochem). Proteins were diluted with SDS-PAGE sample buffer and 1 μg of proteins was electrophoresed on an SDS gel and blotted onto PVDF membrane. The membrane was incubated with 1/10,000 dilution of anti-Xenopus LR antibody. HRP-conjugated secondary antibody was detected by chemiluminescence. For immunohistochemistry, intestine fragments were fixed in 4% paraformaldehyde in 60% PBS at room temperature for 4 hr. Paraffin sections (4 μm) were incubated with 1/4,000 dilution of anti-Xenopus LR antibody in combination with ABC-elite kit (Vector Lab., Burlingame, CA) detected by DAB and counter stained with hematoxylin (Vector Lab.). Total RNA was isolated from various stages of the small intestine using Trizol (Invitrogen). Ten micrograms of total RNA were analyzed by Northern blotting (Amano et al., 2002). 32P labeled Xenopus LR and ST3 were used to hybridize the membrane
Transgenesis and Heat Shock
Transgenesis was done as previously described with a double promoter construct in which the γ-crystalline promoter drives GFP expression in the eyes as a marker and a heat shock inducible promoter drives ST3-GFP expression in the transgenic tadpoles upon heat shock (Fu et al., 2002). F1 animals containing the ST3 transgene under the control of the heat shock inducible promoter were generated through fertilization of F0 transgenic parents. Tadpoles were grown in the same tank until stage 55/56 and the wild type and transgenic animals were heat shocked together. The transgenic and non-transgenic siblings were identified under a UV dissecting microscope based on GFP expression in the eyes of transgenic animals and total proteins were isolated from the intestine and subjected to Western blot analysis as described above.