The prosequence of human lactase-phlorizin hydrolase modulates the folding of the mature enzyme.

The efficient transport of proteins along the secretory pathway requires that the polypeptide adopts a stably folded conformation to egress the endoplasmic reticulum (ER). The transport-competent precursor of the brush border enzyme LPH, pro-LPH, undergoes an intracellular cleavage process in the trans-Golgi network between Arg(734) and Leu(735) to yield LPH beta(initial). The role of the prodomain comprising the N-terminally located 734 amino acids of pro-LPH, LPH alpha, in the folding events of LPH beta(initial) has been analyzed by the individual expression of both forms in COS-1 cells. Following synthesis at 37 degrees C LPH beta(initial) acquires a misfolded and enzymatically inactive conformation that is degraded by trypsin. A temperature shift to 20 degrees C generates a stable, trypsin-resistant, and enzymatically active LPH beta(initial) indicating that the individual expression of LPH beta(initial) results in a temperature-sensitive conformation. This form interacts at non-permissive temperatures sequentially with the ER chaperones immunoglobulin-binding protein and calnexin resulting in an ER retention. The LPH alpha prodomain resides in the ER when individually expressed. It reveals compact structural features that are stabilized by disulfide bridges. LPH alpha and LPH beta(initial) readily interact with each other upon coexpression, and this interaction appears to trigger the formation of a trypsin-resistant, correctly folded, enzymatically active, and transport-competent LPH beta(initial) polypeptide. These data clearly demonstrate that the proregion of pro-LPH is an intramolecular chaperone that is critically essential in facilitating the folding of the intermediate form LPH beta(initial) in the context of the pro-LPH polypeptide.

The efficient transport of proteins along the secretory pathway requires that the polypeptide adopts a stably folded conformation to egress the endoplasmic reticulum (ER). The transport-competent precursor of the brush border enzyme LPH, pro-LPH, undergoes an intracellular cleavage process in the trans-Golgi network between Arg 734 and Leu 735 to yield LPH␤ initial . The role of the prodomain comprising the N-terminally located 734 amino acids of pro-LPH, LPH␣, in the folding events of LPH␤ initial has been analyzed by the individual expression of both forms in COS-1 cells. Following synthesis at 37°C LPH␤ initial acquires a misfolded and enzymatically inactive conformation that is degraded by trypsin. A temperature shift to 20°C generates a stable, trypsin-resistant, and enzymatically active LPH␤ initial indicating that the individual expression of LPH␤ initial results in a temperature-sensitive conformation. This form interacts at non-permissive temperatures sequentially with the ER chaperones immunoglobulin-binding protein and calnexin resulting in an ER retention. The LPH␣ prodomain resides in the ER when individually expressed. It reveals compact structural features that are stabilized by disulfide bridges. LPH␣ and LPH␤ initial readily interact with each other upon coexpression, and this interaction appears to trigger the formation of a trypsin-resistant, correctly folded, enzymatically active, and transport-competent LPH␤ initial polypeptide. These data clearly demonstrate that the proregion of pro-LPH is an intramolecular chaperone that is critically essential in facilitating the folding of the intermediate form LPH␤ initial in the context of the pro-LPH polypeptide.
The endoplasmic reticulum (ER 1 ) is the fabric in which newly synthesized membrane-bound and secretory proteins are extensively processed and shaped to attain a competent mobility within the cell and to achieve a functional maturation. A considerable number of pivotal modification reactions commence already during the translocation of the extending polypeptide across the ER and facilitate ultimately the folding of the protein into a native conformation (for review see Ref. 1). Examples are signal sequence cleavage from the nascent polypeptide chain (2), cotranslational core N-glycosylation at particular Asn residues belonging to the sequon (Asn-X-Ser/ Thr) (3), disulfide bond formation (4), and transient interactions of the folding polypeptide with a set of ER resident accessory proteins, such as molecular chaperones (5,6). In the lumen of the ER, proteins assume their secondary and tertiary structures and, in the case of multisubunit polypeptides, assemble their individual subunits into heterodimeric, homodimeric, or larger order complexes (7). Essentially only correctly folded and assembled proteins can egress the ER and continue their further journey along the secretory pathway. The conformational status of newly synthesized polypeptides is monitored in the lumen of the ER by an efficient quality control mechanism (8). Proteins that fail to acquire a correct three-dimensional structure are retained in the ER and ultimately degraded, presumably in the cytosol by the ER-associated ubiquitin-proteasome pathway (9). Increasing evidence points to the possible existence of a monitoring system that operates beyond the ER. A naturally occurring mutant of intestinal sucrase-isomaltase (10) or a temperature-sensitive mutant of the G protein of vesicular stomatitis virus (11) exit the ER but are blocked in a pre-Golgi compartment instead of transported further to their final destination, the cell surface. The acquisition of a protein to a configuration similar to that of the mature and biologically active species does not always constitute an absolute requirement for its transport along the secretory pathway to the final destination. Nevertheless, retarded transport kinetics and impaired function are frequently the consequences of altered protein folding as has been demonstrated for some mutants of the hemagglutinin of the influenza virus (12) and deletion mutants of intestinal lactase-phlorizin hydrolase (13,14). Presumably, for some proteins only correct folding of particular subdomains is adequate for their mobility along the secretory pathway. It is conceivable that the structural features of the protein critically determine whether or not a protein is retained in the ER by binding specific chaperones if it did not fulfill particular folding criteria.
Several proteins possess proregions or propeptides at the N-terminal end, which undergo cleavage during or after maturation of the precursor polypeptide. Cleavage of these propeptides is associated in many cases with biological activation of the final mature form of the protein (15) as in the cases of zymogens, neuropeptides, and prohormones (16). An important function of propeptides is that of an intramolecular chaperone that regulates and affects the folding of the precursor protein. A well-studied example is subtilisin. The propeptide of this protein comprises 77 amino acids and is critically important for the folding of the remaining 275 amino acids that constitute the mature and active protein (17). Human small intestinal lactase-phlorizin hydrolase (LPH, EC 3.2.1.23-62), an important component of the brush-border membrane, comprises a relatively large proregion (LPH␣) that accounts for about 45% of the precursor molecule (prepro-LPH, 1927 amino acids) (18) and a brush-border mature polypeptide, LPH␤. Initial studies have demonstrated that LPH is synthesized as a single-chain polypeptide precursor, prepro-LPH, that undergoes two sequential intracellular cleavage steps: the first in the ER to pro-LPH (215-kDa) and the second, following terminal glycosylation in the Golgi apparatus, to a 160-kDa LPH (19 -21). Based on sequence analyses of various biosynthetic forms of LPH, recent studies have provided an unequivocal evidence for the existence of an additional cleavage step occurring extracellularly in the intestinal lumen (22,23). Thus, the initial cleavage step of complex-glycosylated pro-LPH takes place at residues Arg 734 /Leu 735 , implicates a trypsin-like protease, and generates an LPH polypeptide encompassing residues 735-1927 (23) that was denoted LPH␤ initial (24). The LPH␤ initial molecule is subsequently targeted to the brush-border membrane where a final digestion by luminal trypsin occurs at Arg 868 /Ala 869 , which eliminates a peptide stretch from residues Leu 735 to Arg 868 yielding LPH␤ final (22,23). This form is the polypeptide that exerts its enzymatic function as a ␤-galactosidase in the small intestine. The cleavage of pro-LPH to LPH␤ initial and its potential implications on the function and trafficking on this form has been studied in heterologous transfection systems. Meanwhile it has become clear that uncleaved pro-LPH is a transport-competent (25), a correctly sorted (26,27), and an enzymatically active molecule (25), and hence cleavage of pro-LPH in intestinal cells is neither required for activation of the enzyme, nor is it implicated in the transport and sorting events. LPH␤ initial contains all the information needed for apical sorting of pro-LPH, whereas LPH␣ is devoid of sorting signals despite striking sequence homologies shared with LPH␤ initial (18). Moreover, the LPH␣ profragment is devoid of catalytic activity, because the lactase and phlorizin hydrolase activities have been assigned to glutamates 1271 and 1747 of LPH␤ final , respectively (28), and, moreover, the activities of the precursor pro-LPH and LPH␤ final are identical (25) for lactose or phlorizin hydrolysis.
Based on the initial concepts that pro-LPH is immediately cleaved at the Arg 868 /Ala 869 site a cDNA clone encoding the polypeptide Ala 869 -Phe 1927 , LPH␤ final , was expressed individually in the absence of the profragment, and its biosynthetic and structural features were analyzed. It was shown that the polypeptide generated was an enzymatically inactive protein, and most of it was retained as a mannose-rich glycosylated indicative of a predominant ER localization. This has lead to the hypothesis that the profragment LPH␣ functions as an intramolecular chaperone that is implicated in the folding of the LPH␤ domain in the ER (29,30). The intestinal form of LPH␣ is neither N-nor O-glycosylated, despite the presence of five potential N-glycosylation sites, and is rich in cysteine and hydrophobic amino acid residues. These features have suggested that LPH␣ folds rapidly into a tight and rigid globular domain in which carbohydrate attachment sites are no longer accessible to glycosyltransferases. In this paper we investigate the folding features of LPH␤ and LPH␤ initial and the putative role of LPH␣ as an intramolecular chaperone. Additionally, in view of the new constellation that the initial cleavage occurs at Arg 734 /Leu 735 rather than at Arg 868 -Ala 869 , it became important to determine the role of the polypeptide stretch Leu 735 -Arg 868 in the processing and folding within the pro-LPH species.
Construction of cDNA Clones-LPH␤ initial was cloned in two steps. First, the cDNA encoding the signal sequence of pro-LPH, LPH signal , was amplified by PCR as described before (30). A second part of the cDNA comprising the last 20 nucleotides of the LPH signal sequence (nucleotides 52-71) and nucleotides 2203-3572 of LPH␤ initial was synthesized by PCR using LPHcDNA as template and the oligonucleotides LPHsig/beta (GTT TTT CAT GCT GGG GGT CAC TGT TGC AGT TTG TAT CCC T) and cLPH3600 (25). Both DNA fragments were then fused by assembly PCR and cloned with the 3Ј-EcoRI/HindIII fragment of LPH cDNA into the unique EcoRI site of the pJB20 expression vector (27) by a three-way ligation. The resulting construct denoted pJB20-LPH␤ initial was sequenced and found to contain the signal sequence of pro-LPH fused in-frame to the cDNA beginning with nucleotide 2203. For the generation of a CFP fusion protein the EcoRI/ScaI fragment of pJB20-LPH␤ initial was subcloned into the EcoRI/SmaI-digested pECFP-N1 vector (CLONTECH, Inc., Heidelberg, Germany) to generate pLPH␤ initial -CFP.
The profragment of pro-LPH, LPH␣, extending to nucleotide 2202 was first amplified from the LPHcDNA template by PCR with the primer pair LPH1 (25)/cLPH␣ (AAT CTA GAG CCT CCT TAT CCC CCA G), which inserts a stop codon at position 2203 in the LPH␣cDNA. This DNA fragment was inserted into the unique EcoRI/XbaI sites of pcDNA3 (Invitrogen, Groningen, The Netherlands). The resulting construct, pcDNA3-LPH␣, was confirmed by sequencing. LPH␣ and YFP were fused by PCR amplification of LPH␣ using full-length LPHcDNA as template with the primer pair LPH1HindIII (AAA AGC TTC CTA GAA AAT GGA GCT G)/cLPH␣ SalI (CCT CGT CGA CCT CCT TAT CCC CCA GGG) and ligation of the PCR product into the HindIII/SalI sites of pEYFP-N1. The generated plasmid was confirmed by sequencing and named pLPH␣-YFP.
Transient Transfection of COS-1 Cells, Biosynthetic Labeling, and Immunoprecipitation-COS-1 cells were transiently transfected with DNA by using DEAE-dextran essentially as described previously (25). pJB20-LPH␤ initial , pcDNA3-LPH␣, or a mixture of both plasmids, pLPH␣-YFP and pLPH␤ initial -CFP, were used. 48 h after transfection, the cells were biosynthetically labeled. Here, the cells were preincubated with 5 ml of methionine-free MEM for 1 h after which time the medium was changed and replaced by a similar medium containing 50 Ci of [ 35 S]methionine. Labeling was performed for 1 h followed by a chase with non-labeled methionine for 4 h. After labeling, the cells were rinsed twice with cold phosphate-buffered saline and solubilized with 1 ml of lysis buffer containing 25 mM Tris-HCl (pH 8.0), 50 mM NaCl, 0.5% sodium deoxycholate, and 0.5% TX-100 supplemented with 1 mM phenylmethylsulfonyl fluoride, 1 g/ml pepstatin, 5 g/ml leupeptin, 5 g/ml aprotinin, 1 g/ml antipain, and 50 g/ml trypsin inhibitor for 30 min at 4°C. The cell extracts were centrifuged to remove cell debris while the supernatant was retained for the subsequent immunoprecipitation. After 1 h of preclearing with 20 l of protein A-Sepharose, the immunoprecipitation was performed with polyclonal anti-LPH␣ (V496) or a mixture of mAb anti-LPH (MLac 2, MLac 6, and MLac 8) and 50 l of protein A-Sepharose as described previously (13). For assessment of the interaction of the various LPH␤ initial forms with BiP or calnexin, the cellular extracts were immunoprecipitated with mAb anti-BiP or mAb anti-calnexin (32). The immunoprecipitates were eluted by boiling in 1% SDS for 10 min followed by 10-fold dilution of SDS using a buffer containing 1% Triton X-100. Thereafter the samples were immunoprecipitated with a mixture of anti-LPH antibodies directed against native and denatured forms of the protein. The immunoprecipitates were further processed on SDS-PAGE.
Trypsin Treatment of Cell Lysates-LPH␤ initial was immunoprecipitated from transient transfected COS-1 cells labeled with [ 35 S]methionine for 6 h. The immunoprecipitates were treated with 50 g/ml trypsin for the indicated times at 37°C. The reaction was stopped by the addition of 2 mg/ml soybean trypsin inhibitor (Roche Molecular Biochemicals, Mannheim, Germany).
Cell-free Transcription/Translation-The vector pcDNA3-LPH␣ containing the profragment of human pro-LPH was linearized with ApaI. Transcription was initiated with T7-RNA polymerase and carried out essentially as described before (32). Subsequently, the mixture was extracted once with phenol/chloroform and twice with chloroform. After ethanol precipitation, the RNA was resuspended in 50 l of RNase-free water. Translation of the newly synthesized RNA was performed in nuclease-treated rabbit reticulocyte lysate. The reaction mixture comprised 18 l of reticulocyte lysate, 1 l of 1 mM amino acids (minus methionine), 15 Ci of L-[ 35 S]methionine, and 3 l of transcribed RNA. Where indicated, the samples were supplemented with 1 l of nucleasetreated microsomal membranes. Translations were performed in the absence or presence of 2 or 6 mM oxidized glutathione (GSSG) at 30°C for 60 min. Microsomal membranes were isolated as described by Bulleid and Freedman (33). Samples were prepared for electrophoresis by mixing with 5 vol. of SDS-PAGE sample buffer (62.5 mM Tris-HCl, pH 6.8, SDS (2% w/v), glycerol (10% w/v), and bromphenol blue) in the presence of 50 mM dithiothreitol for reducing conditions and for nonreducing conditions in the presence of 100 mM iodoacetamide and boiled for 5 min. After electrophoresis, gels were processed for autoradiography and exposed to Kodak X-Omat AR film.
Confocal Fluorescence Microscopy-Confocal images of living cells were acquired 2 days after transfection on a Leica TCS SP2 microscope with a ϫ63 water planapochromat lens (Leica Microsystems) (34). Dual color CFP and YFP images were obtained by sequential scans with the 458-and 514-nm excitation lines of an argon laser and the optimal emission wavelength for CFP or YFP, respectively.
Enzymatic Assays-Lactase activity was measured according to Dahlqvist (35) using lactose as substrate. LPH␤ initial was immunoprecipitated from cell lysates of transiently transfected COS-1 cells. For each determination of the lactase activity, the lysate equivalent to fourteen 100-mm-diameter dishes of confluent cells was utilized. The immunoprecipitates were subsequently assayed for lactase activity essentially as described (25).

RESULTS
The LPH␤ initial Polypeptide Is a Temperature-sensitive Folding Mutant-The cDNA encoding the initial cleavage product, LPH␤ initial (Leu 735 -Phe 1927 ) was fused to the signal sequence of pro-LPH to allow translocation across the ER membrane ( Fig.  1). Expression of this hybrid form in COS-1 cells and assessment of its glycosylation pattern using endo H sensitivity revealed a strongly labeled 150-kDa mannose-rich polypeptide (LPH␤ initial/h ) in addition to a 175-kDa endo H-resistant complex protein (LPH␤ initial/c ). The proportions of these two forms at steady state demonstrated a predominant presence of the mannose-rich protein with ϳ70% of the total LPH␤ initial protein. When LPH␤ final was expressed individually the amount of complex-glycosylated LPH␤ final was substantially reduced (30) suggesting a role of the Leu 735 -Arg 866 stretch in the processing of LPH␤ initial . Nevertheless, the processing of mannose-rich LPH␤ initial (LPH␤ initial/h ) to the complex-glycosylated form (LPH␤ initial/c ) was markedly less efficient in comparison with that of wild type pro-LPH ( Fig. 2A) pointing to an altered folding pattern of LPH␤ in the absence of the profragment. The enzymatic activity of lactase has been assigned to residues Glu 1273 and Glu 1749 within the LPH␤ initial domain (36). Individual expression of this domain is associated with a drastic reduction in the lactase activity (Table I) reminiscent of a misfolded structure of LPH␤ initial at least at or in the vicinity of the catalytic site. However, the partial conversion of LPH␤ initial from a mannose-rich polypeptide into a complex-glycosylated protein suggests that a proportion of the LPH␤ initial protein has acquired a correct conformation albeit at a slower rate. Alternatively, minimal folding requirements of LPH␤ initial were fulfilled permitting the partially folded protein to egress the ER. We wanted therefore to determine whether slowing down the processing rate of LPH␤ initial and prolonging its residence time in the ER influences the structural features of LPH␤ initial and its biological activity. For this purpose, cells transfected with the cDNA encoding LPH␤ initial were labeled biosynthetically at 20°C. Fig. 2B shows that the mannose-rich polypeptide was converted to a complex-glycosylated protein after 8 h of chase. Interestingly, this protein was now as enzymatically active as its wild type pro-LPH counterpart (Ref. 25 and Table I). We FIG. 1. Schematic representation of the structure of pro-LPH in human small intestinal cells and the two constructs LPH␤ initial and LPH␣. Some important structural features of pro-LPH in human small intestinal cells. Shown is the cleavable sequence (Met 1 -Gly 19 ) at the N terminus. The ectodomain encompasses amino acid residues Ser 20 to Thr 1882 . The intracellular proteolytic cleavage takes place between Arg 734 and Leu 735 to generate LPH␤ initial . The luminal extracellular cleavage occurs at Arg 868 to generate the brush-border mature enzyme, LPH␤ final (Arg 868 -Phe 1927 ) (22,23). The construct LPH␤ initial was generated by fusing the 19 amino acids of the LPH signal sequence to Leu 735 , the N terminus of LPH␤ initial . For expression of LPH␣, a stop codon was introduced after Arg 734 . MA, membrane anchoring; CT, cytoplasmic tail.
next compared the fate of LPH␤ initial under the two different labeling temperatures by employing a pulse-chase protocol.
Here, a dramatic difference in the turnover rates was observed. Although LPH␤ initial was gradually degraded with increasing chase periods at 37°C, longer chase periods of up to 24 h at 20°C have resulted in an efficient conversion of mannose-rich LPH␤ initial/h to a meanwhile predominantly labeled complexglycosylated LPH␤ initial/c . This increase in stability of LPH␤ initial after synthesis at 20°C was also observed by determination of the steady-state level of the polypeptide after synthesis at 37°C or 20°C in a Coomassie Blue-stained polyacrylamide gel (Fig. 2C). In these experiments LPH␤ initial/h was isolated from transfected COS cells incubated at 37°C, whereas LPH␤ initial/c was detected in cells cultured at 20°C. This indicates that reduced temperatures facilitate the formation of a stable conformation of complex-glycosylated LPH␤ initial/c . A direct comparison of the forms generated at the two different temperatures reveals an increase of ϳ10 kDa in the apparent molecular mass of LPH␤ initial/c formed at 37°C as compared with the isoform at 20°C (Fig. 3A). One possible explanation for this difference could be altered sugar contents in both forms. To determine this content, pulse-chase experiments, with transfected cells at 37°C or 20°C combined with deglycoslyation of the LPH␤ initial isoforms with neuraminidase and endo F/GF, were performed (Fig. 3A). At 4 h of chase the mannose-rich glycosylated LPH␤ initial/h was immunoprecipitated from the cell lysates, which was sensitive to endo F/GF and expectedly devoid of sialic acid as assessed by its insensitivity to neuraminidase treatment. Within 12 h of chase complex-glycosylated LPH␤ initial/c appeared and shifted to 155 kDa after desialylation with neuraminidase. Neuraminidase also reduces LPH␤ initial/c synthesized at 37°C to the 155-kDa polypeptide. Therefore, the difference in the apparent molecular masses of the LPH␤ initial at both temperatures is due to a variable content of terminal sialic acid residues. To analyze if this difference in glycosylation of LPH␤ initial/c has any influence on or is related to changes in the folding and stability of the protein, we probed its sensitivity toward trypsin. Correctly folded wild type brush-border LPH␤ is resistant to trypsin treatment (25). Changes in the protein folding of LPH␤ initial would be therefore monitored by variation in its susceptibility to trypsin. As shown in Fig. 3B, trypsin resulted in a drastic degradation of LPH␤ initial/h and LPH␤ initial/c , which were synthesized at 37°C.

FIG. 2. Biosynthesis and transport kinetics of LPH␤ initial in COS-1 cells.
A, COS-1 cells were transiently transfected with pJB20-LPH␤ initial , biosynthetically labeled for 6 h followed by immunoprecipitation of LPH␤ initial . The immunoprecipitates were divided into three aliquots and treated with endo H, endo F/GF, or not treated. The proteins were subjected to SDS-PAGE and autoradiography. B, transiently transfected COS-1 cells were pulsed with [ 35 S]methionine for 1 h at 37°C followed by different chase times at 20°C or 37°C. Cells were lysed, and the immunoprecipitates were analyzed by SDS-PAGE and autoradiography. C, immunoprecipitates of transiently transfected COS-1 cells incubated at 20°C or 37°C were loaded onto SDS-PAGE followed by Coomassie Blue staining of the gel.

TABLE I
Enzymatic activity of LPH␤ initial in transfected COS cells COS-1 cells were transiently transfected with pJB20-LPH␤ initial alone or in combination with pcDNA3-LPH␣ and incubated at 37°C for 48 h. Following cell lysis LPH␤ initial was immunoprecipitated and the lactase activities in the immunoprecipitates were measured according to Dahlqvist (35). Results are the mean Ϯ S.E. of three experiments.
FIG. 3. Processing and folding of LPH␤ initial at 20°C. A, COS-1 cells were transiently transfected with pJB20-LPH␤ initial , biosynthetically labeled at 20°C for 4 h, and chased for the indicated times or labeled at 37°C with [ 35 S]methionine for 2 h followed by a 12-h chase. After cell lysis LPH␤ initial was immunoprecipitated and the immunoprecipitates were divided into equal aliquots and treated with endo F/GF, neuraminidase, or not treated. The proteins were subjected to SDS-PAGE and autoradiography. B, trypsin sensitivity assay of LPH␤ initial . Transiently transfected COS-1 cells were biosynthetically labeled at 37°C for 8 h or at 20°C for 12 h followed by immunoprecipitation of LPH␤ initial from the cell lysates. The immunoprecipitates were treated with trypsin for different times and analyzed on SDS-PAGE. The trypsin-sensitive form of LPH␤ initial/c detected after synthesis at 37°C is indicated by asterisks.
After 5 min of incubation with trypsin, almost all of the LPH␤ initial was degraded. By contrast, LPH␤ initial synthesized and processed at 20°C was resistant to trypsin. It is worthwhile to note that a minor proportion of LPH␤ initial synthesized at 37°C was resistant to trypsin, and, interestingly enough, this proportion had a similar apparent molecular mass as the correctly folded LPH␤ initial synthesized at 20°C. The acquisition of trypsin resistance at 20°C similar to the wild type enzyme strongly suggests that LPH␤ initial exhibits characteristics of a temperature-sensitive protein in the absence of the LPH␣ profragment. The variation in the folding pattern observed at 37°C is also associated with an altered terminal glycosylation pattern.
LPH␣ Alone Is a Compact Globular Polypeptide-Obviously the presence of the LPH␣ profragment is crucial in influencing the folding of LPH␣ initial in the context of wild type pro-LPH. It has been previously postulated that the LPH␣ molecule, which contains 11 cysteine residues, attains a compact structure that is stabilized by disulfide bridges and that acts as a kernel for the folding of the mature enzyme (30). To characterize the role of this domain in this context, we assessed its structural features in an in vitro translation assay. The cDNA encoding LPH␣ was transcribed in vitro and translated with rabbit reticulocyte lysate supplemented with dog pancreatic microsomal vesicles. This system has already been used to demonstrate the presence of disulfide bridges in the full-length pro-LPH enzyme (32). The main polypeptide synthesized in the absence of added microsomal vesicles had an apparent molecular mass of 90 kDa (Fig. 4A) consistent with the molecular weight of LPH␣ calculated from the deduced amino acid sequence (18). In the presence of microsomal membranes an additional polypeptide of 100 kDa was generated. Separation of the microsomal membranes from the reticulocyte lysates by centrifugation through a sucrose cushion revealed a predominant labeling of this species in the microsomal membranes. The 100-kDa protein is mannose-rich and glycosylated, because it was sensitive to treatment with endo H and converted to the nascent unglycosylated 90-kDa polypeptide. The nascent 90-kDa translation product was therefore exposed to the glycosylation machinery in the lumen of the microsomal membranes. To investigate the presence of cotranslationally formed disulfide bonds, the translation reaction was carried out in the presence of oxidized glutathione (GSSG). GSSG oxidizes existing thiol groups and facilitates, therefore, the formation of disulfide bonds. It is required in the reaction mixture to compensate for the presence of the reducing agent dithiothreitol in the commercially prepared reticulocyte lysates. 2 or 6 mM GSSG was added to the reticulocyte lysates before initiation of translation to obtain a lysate that was competent in forming disulfide bonds in the newly synthesized protein. A change in the thiol/disulfide redox status of the translation products has been shown to influence the migration pattern of many proteins (37). In the absence of GSSG and under non-reducing conditions, both the 90-kDa as well as the glycosylated 100-kDa polypeptides could be detected (Fig. 4B). On the other hand, the addition of GSSG to the translation mixture results in a substantial increase in the mobility of the microsomal form of LPH␣ from a 100-kDa species to one of ϳ70 kDa under non-reducing conditions. The faster migration behavior of LPH␣ through the gel matrix is reminiscent of a compact conformation of this form facilitated by the formation of intramolecular disulfide bonds in the presence of GSSG. The primary sequence of LPH␣ contains 11 cysteine residues as putative candidates for the formation of disulfide bridges, and the obvious mobility shift observed implies that more than one disulfide bond might be formed to reach that high degree of condensation.
LPH␣ Resides in the ER of Transfected COS-1 Cells-We further analyzed the structural features of LPH␣ in vivo after expression in COS-1 cells. LPH␣ was immunoprecipitated from biosynthetically labeled transfected cells with the polyclonal antibody V496, which binds to the N-terminal part of LPH␣ (30), followed by treatment of the immunoprecipitates with endo H or endo F/GF. Fig. 5A shows that three forms of LPH␣ were revealed. A major 100-kDa component similar to the N-glycosylated polypeptide was synthesized in the cell-free system and two minor 103-and 97-kDa polypeptides. All three forms were converted to the non-glycosylated 90-kDa species upon N-deglycosylation with either endo H or endo F/GF, indicating that they correspond to various glycosylated isoforms of the same polypeptide. Furthermore, by virtue of the specificity of endo H in cleaving mannose-rich N-glycans and endo F/GF in cleaving mannose-rich as well as complex N-linked glycans, the data indicate that LPH␣ is exclusively mannose-rich and glycosylated and is consequently located in the ER and is not further transported to the Golgi apparatus.
These findings were corroborated by confocal microscopic analyses of COS-1 cells expressing the yellow fluorescence protein (YFP) fused to LPH␣ (LPH␣-YFP). The transfected cells revealed exclusive labeling of the ER (Fig. 5B).

LPH␤ initial Polypeptide Acquires Enzymatic Activity and Trypsin-resistant Conformation in the Presence of LPH␣-Hav-
ing assessed the structural features of LPH␤ initial and LPH␣ individually we wanted next to examine directly the role of LPH␣ on the folding of LPH␤ initial . One approach is to examine whether the enzymatic activity of LPH␤ initial increases in the presence of LPH␣ reminiscent of acquisition of correct folding. For this, COS-1 cells were cotransfected with the cDNAs encoding LPH␤ initial and LPH␣. In the control cells LPH␤ initial was individually expressed. As shown above, the enzymatic activity of LPH␤ initial expressed alone in cells cultured at 37°C was very low, whereas this protein was enzymatically active only at the permissive temperature of 20°C (Table I). On the other hand, the cotransfected cells reveal a dramatic elevation in the enzymatic activity of LPH␤ initial .
In the second approach the conformation of LPH␤ initial in the presence or absence of LPH␣ was probed with trypsin to determine whether alterations in the folding pattern have occurred. LPH␤ initial was expressed in COS cells alone or in the presence of LPH␣. The biosynthetically labeled proteins were immunoprecipitated with mAb anti-LPH and treated with trypsin or with V496, the polyclonal anti-LPH␣ antibody. Fig. 6 shows that in the absence of cotransfected LPH␣ almost half of the LPH␤ initial was cleaved by trypsin to a smaller polypeptide pair corresponding to the mannose-rich and complex-glycosylated forms. Immunoprecipitation of the cellular lysates with V469, the anti-LPH␣ antibody, was utilized as an internal control to confirm the absence of this protein in this experimental set up. On the other hand, coexpression of LPH␤ initial with LPH␣ leads to a substantial decrease in the proportion of LPH␤ initial cleaved by trypsin to less than 10% reminiscent of an increased stability of this protein. The presence of the LPH␣ protein was confirmed by it reactivity with the V496 antibody. It is established that the 160-kDa wild type brush-border LPH␤ is trypsin-resistant (25). That a complete resistance of LPH␤ initial to trypsin has not been achieved is most likely due to the heterogeneous transfected cell populations, which contained both genes, or the individual ones, and therefore an optimal effect on LPH␤ initial could be attained. This is also the reason why the enzymatic activity could not be fully restored to normal brushborder levels.
Finally, a significantly higher proportion of the complexglycosylated species in the total synthesized LPH␤ initial protein was detected in presence of LPH␣ as compared with that of the individually expressed LPH␤ initial (69.8% versus 79.2%). This increase suggests a more efficient processing and transport rate of mannose-rich LPH␤ initial as a consequence of altered folding characteristics in the presence of LPH␣.
In conclusion, LPH␤ initial is a temperature-sensitive protein that is characterized by an improperly folded, trypsin-sensitive, and biologically inactive protein at 37°C. Normal structural and functional features could be restored either at 20°C or by addition of the prodomain LPH␣, indicating that these N-terminal 734 amino acids of the proregion would assist the folding of LPH␤ initial at 37°C.

LPH␣ Is Associated with LPH␤ initial When Expressed in
COS-1 Cells-Having assessed the role of LPH␣ on LPH␤ initial , we wanted to determine how this effect ensues and whether there is a direct interaction between the two proteins in the ER, where the most crucial folding events take place. For this, LPH␤ initial and LPH␣ were coexpressed in COS-1 cells and each protein was immunoprecipitated from the detergent extracts of biosynthetically labeled cells, either with mAb anti-LPH antibody, directed against LPH␤ initial , or pAb V496 antibody directed against the LPH␣. Fig. 7A demonstrates that both species coimmunoprecipitated with either antibody despite the specific affinity of the antibodies used toward a particular epitope found on the individual subunits and not on both. The question that arises is whether the association between both subunits of pro-LPH results in the attainment of a native conformation of LPH␤ initial . This is indeed the case, because there is an increase in the proportion of LPH␤ initial/c due to the presence of LPH␣ in trans and its direct association after biosynthesis with LPH␤ initial . The low amount of LPH␣ coprecipitating with LPH␤ is compatible with a transient interaction occurring between these two forms that is terminated as soon as the LPH␤ protein has acquired a correct conformation. Moreover, this interaction appears to be specific to LPH, because the LPH␣ prosequence is not capable of modulating the folding of the isomaltase or sucrase subunits of the sucraseisomaltase enzyme complex. 2 Neither the isomaltase nor the sucrase subunits are correctly folded species when they are individually expressed, and they require each other to be transported along the secretory pathway. Here the presence of the LPH␣ could not substitute for the role of either subunit in the folding process of the other.
To visualize the effect of LPH␣ on LPH␤ in life cells, the two proteins were fused either to the cyan fluorescent protein, CFP (LPH␤ initial -CFP), or the yellow fluorescent protein, YFP 2 R. Jacob, B. Pü rschel, and H. Y. Naim, manuscript in preparation. . (LPH␣-YFP). Confocal microscope imaging of transfected COS-1 cells revealed a predominant ER localization of the LPH␤ initial -CFP (Fig. 7B, panel a). On the other hand, when both subunits, LPH␣-YFP and LPH␤ initial -CFP, were coexpressed in the same cell, LPH␤ initial -CFP could be localized in the Golgi apparatus, in transport vesicles and also at the cell surface (Fig. 6B, panels b-d). Here, both species were expressed at virtually similar levels as assessed by quantification of the specific fluorescence, excluding an imbalanced expression of the subunits. In the absence of LPH␣-YFP no significant labeling of LPH␤ initial -CFP in the Golgi or at the cell surface could be observed (Fig. 7B, panel a). Together, these observations support the biochemical data, which showed that the individual expression of LPH␤ results in a smaller proportion of mature and complex-glycosylated LPH␤ as compared with wild type pro-LPH and LPH␤ coexpressed with LPH␣ (see Table II, Fig. 6, and Ref. 30).
The ER-resident Molecular Chaperones BiP and Calnexin Assist the Folding of LPH␤-The trans effect of LPH␣ on the folding of LPH␤ initial has lead us to ask how this effect would associate and conform with other folding events that implicate ER components, such as BiP and calnexin (38). The putative and temporal interaction of LPH␤ initial with BiP and calnexin was analyzed in transiently transfected COS-1 cells either expressing LPH␤ initial alone or in addition of LPH␣. A pulsechase analysis of these cells followed by coprecipitation of chaperone-bound LPH␤ initial with anti-BiP and anti-calnexin antibodies is demonstrated in Fig. 8. After a pulse period of 10 min the mannose-rich polypeptide of individually expressed LPH␤ initial (i.e. in the absence of LPH␣) could be found associated with BiP, but not calnexin. Essentially a reversed binding pattern was obtained within 30 min of chase. Here, LPH␤ initial bound to calnexin and only slightly to BiP. The same pattern was also revealed after 60 min of chase. Later, at 120 min of chase, however, the earliest binding profiles of LPH␤ initial to the two chaperones were repeated, i.e. strong binding to BiP and almost negligible or no binding to calnexin. Obviously LPH␤ initial is subject to several cycles of association, dissociation, and reassociation with ER-resident proteins. Each of these cycles commences with the binding of BiP to an early folding intermediate of LPH␤ initial followed by its dissociation and the association with calnexin, which in turn dissociates followed by the reassociation of BiP. The prolonged period of binding to these two chaperones to LPH␤ initial at virtually similar intensities throughout is presumably due to an inefficient folding of LPH␤ initial .
The sequential pattern of association of LPH␤ initial with BiP and calnexin changes substantially when LPH␤ initial was coexpressed with LPH␣. Here, only BiP, but not calnexin, bound to LPH␤ initial . The labeling intensity of LPH␤ initial that was associated with BiP decreased concomitantly with the appearance of complex-glycosylated forms (Fig. 8). In sum, the data demonstrate that the absence of the LPH␣ prodomain exposes binding sites for calnexin resulting in the retention of partially folded forms of LPH␤ initial by this chaperone and a repeated association with BiP. LPH␣, on the other hand, assists the folding of LPH␤ initial to a transport-competent form that leaves the ER for further transport to the cell surface. At least in part, calnexin compensates for the absence of LPH␣ by retaining LPH␤ initial until BiP binds and the protein goes into a new folding cycle. DISCUSSION The initial intracellular proteolytic cleavage of intestinal pro-LPH in the trans-Golgi network at Arg 734 /Leu 735 (22) is a major processing event along the secretory pathway of this protein. The generated LPH␤ initial is sorted to the apical membrane, where it undergoes another cleavage step by trypsin to generate the brush-border LPH protein, LPH␤ final (22,23). Two independent studies have previously provided strong evidence that individual expression of sequences encoding brush-border LPH, i.e. Arg 867 -Phe 1927 , result in a transport-incompetent and enzymatically inactive protein (29,30) thus implicating the profragment in the folding events of pro-LPH as an intramolecular chaperone.
In the present report we addressed the putative role of LPH␣ as an intramolecular chaperone as well as the interplay between this domain, the LPH␤ initial , and two ERresident proteins. These data demonstrate that LPH␤ initial is a temperature-sensitive protein that folds properly to an active protein at 20°C but is misfolded and inactive under the physiological temperature. The first folding steps of proteins destined for transport along the secretory pathway occur in the ER, which harbors a folding machinery comprising a battery of molecular chaperones. Two members of this machinery, BiP and calnexin, interact with LPH␤ initial at the non-permissive temperature, whereby this interaction follows a sequential pattern and most likely proceeds for several cycles. The cycles of association, dissociation, and reassociation with the chaperone and the longer residence of the LPH␤ initial protein in the ER are apparently not sufficient for the attainment of the protein to a mature, transportcompetent configuration. In fact, only a minor proportion of this protein egresses the ER and acquires a complex type of glycosylation in the Golgi. Despite traversing the quality control machinery in the ER, these polypeptides are enzymatically inactive and sensitive toward trypsin treatment. This is reminiscent of altered folding that is also reflected by a variation in the N-glycosylation pattern, particularly that of the sialic acid content, as compared with the correctly folded protein. A dual function of molecular chaperones in regulating the folding of proteins has been described for many proteins. In contrast to LPH␤ initial , however, the proteins in  8. Sequential interaction of LPH␤ initial with BiP and calnexin. COS-1 cells were transfected with a combination of pcDNA3-LPH␣ and pJB20-LPH␤ initial or pJB20-LPH␤ initial . 48 h post transfection, the cells were labeled for 10 min followed by different chase intervals. The cell lysates were immunoprecipitated with mAb anti-LPH and when indicated with anti-BiP or anti-calnexin. The samples were analyzed on SDS-PAGE and autoradiography. these cases acquire ultimately a correctly folded configuration. For example, the folding of the G protein of the vesicular stomatitis virus is critically dependent on the interaction of this protein with BiP and calnexin (39), whereas the immunoglobulin chains interact with BiP and GRP94 (40). The assembly of the multitransmembrane domains of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in the cytosol is facilitated through the sequential binding with the chaperones hdj-2 and hsc-70 (41), and in the case of thyroglobulin the interaction with molecular chaperones follows a precursor-product relationship (42).
The defect in the folding pattern of LPH␤ initial at the nonpermissive temperature can be only overcome when the Nterminally located LPH␣ is present. Here, enzymatic activity as well as correct folding is restored. One of the early examples of prodomains with a folding function as an intramolecular chaperone is the prodomain of subtilisin (15,43). After maturation of the precursor molecule, this polypeptide is proteolytically cleaved to generate the biologically active protease. It can guide the folding of the inactive protein to an active protease in vitro when added exogenously (17). Intramolecular chaperone function has also been described for the prodomains of activin A (44), transforming growth factor ␤1 (44), cathepsin C (45), and type 1 matrix metalloproteinase (46). The 13.5-kDa N-terminal part of cathepsin C folds spontaneously and rapidly to a compact monomer with stable tertiary interactions (45). It is involved in the sequential formation of disulfide linkages in the native protein. This has also been observed for the 13-residue proregion of bovine pancreatic trypsin inhibitor, which provides an intramolecular thiol disulfide reagent capable of assisting and accelerating disulfide bond formation in the native enzyme (47). In an oxidizing milieu such as that of the ER the LPH␣ domain assumes a compact configuration stabilized by disulfide bridges that link several of the 11 cysteine residues (see "Results") 3 and facilitated by hydrophobic interaction of almost 45% of uncharged, non-polar amino acid residues that are likely to be oriented into the interior space of this protein.
These structural features are favored by the electrophoretic behavior of the faster migrating oxidized LPH␣ on SDS gels as compared with the non-oxidized isoform as well as the reduced N-glycosylation. Potential N-linked glycosylation sites in proteins are usually more accessible to glycosylation in unfolded proteins than their folded and compact counterparts (48). In analogy with other profragments and by virtue of its decisive function in the folding of LPH␤ initial one would assume that LPH␣ facilitates the accurate formation of disulfide bridges in the LPH␤ initial domain, which are indispensable for the correct folding and generation of a native pro-LPH (32). Moreover, the direct binding of LPH␣ to LPH␤ initial , as shown in the in vitro assays, suggests that LPH␣ could function to prevent misfolding, to permit an autonomous folding of LPH␤ initial , and to help generate disulfide bonds in the ER. It should be noted that the interaction of the LPH␣ prosequence is most likely specific to LPH, because it is not capable of modulating the folding of the isomaltase subunit of the sucrase-isomaltase enzyme complex. Here, neither the isomaltase nor the sucrase subunits are competently transported along the secretory pathway when they are individually expressed.
By virtue of the striking sequence similarities between LPH␣ and LPH␤ initial it is likely that LPH␣ builds a kernel structure immediately after synthesis that functions as a folding template for other homologous structural domains of LPH␤ initial . This would also explain why several cycles of sequential interaction of LPH␤ initial with the two chaperones, BiP and calnexin, in the absence of LPH␣ are not sufficient for LPH␤ initial to acquire correctly folded confirmation whereas the binding of LPH␣ was.
Current concepts suggest that at least two additional mechanisms specify the function of intramolecular chaperones, an unfolding of misfolded conformations, and a nonspecific prevention of protein misfolding. The mode of action of LPH␣ on the folding of LPH␤ initial provides an additional novel mechanism of this type of domains.
The breakage of the cycles of sequential binding of LPH␤ initial to BiP and calnexin, in the presence of LPH␣, prevent the interaction of LPH␤ initial with calnexin, suggesting that both polypeptides, LPH␣ and calnexin, compete in binding to common sites on LPH␤ initial . Nevertheless, calnexin by itself is not able to replace LPH␣, because it does not share homologies with LPH␤ initial as LPH␣ does and is therefore unable to behave as a folding template for LPH␤ initial .