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(Journal of Leukocyte Biology. 2000;68:669-678.)
© 2000 by Society for Leukocyte Biology

Structural requirements for intracellular processing and sorting of bactericidal/permeability-increasing protein (BPI): comparison with lipopolysaccharide-binding protein

Elinor Bülow, Urban Gullberg and Inge Olsson

Department of Hematology, Research Department 2, University Hospital, Lund, Sweden

Correspondence: Elinor Bülow, M.D., Research Department 2, E-blocket, University Hospital, S-221 85 Lund, Sweden. E-mail: elinor.bulow{at}hematologi.lu.se


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The bactericidal/permeability-increasing protein (BPI), which is stored in the azurophil granules of neutrophils, and the circulating lipopolysaccharide-binding protein (LBP) share the same structure. Both bind lipopolysaccharide of gram-negative bacteria through their amino-terminal domains. The carboxy-terminal domain of BPI promotes bacterial attachment to phagocytes, whereas the corresponding domain of LBP delivers lipopolysaccharide to monocytes/macrophages. Our aim was to investigate the role of the amino- and carboxy-terminal domains of BPI and LBP for sorting and storage in myeloid cells after transfection of cDNA to two rodent hematopoietic cell lines. Full-length BPI and LBP were both targeted for storage in these cells. Deletion of the carboxy-terminal half of BPI resulted in storage followed by degradation while the reciprocal deletion of the amino-terminal half led to retention in the endoplasmic reticulum for proteasomal degradation. Chimeras between halves of BPI and LBP were also targeted for storage, but those containing carboxy-terminal BPI had the highest stability, again indicating a role for the carboxy-terminal domain of BPI in protection against degradation. Therefore, we propose a critical stability function for the hydrophobic carboxy-terminal domain of BPI during intracellular sorting for storage while the amino-terminal domain may confer targeting for storage.

Key Words: azurophil granule • chimera • neutrophil • secretion • stability


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophil granulocytes play an essential role in the innate immunity by generating oxygen radicals as well as by mobilizing antibiotic proteins and peptides. Among the antibiotic proteins is a cationic protein named bactericidal/permeability-increasing protein (BPI) with a pivotal role in host defense against gram-negative bacteria [1 2 3 4 ]. The intracellular processing in myeloid cells of BPI and its circulating homolog the lipopolysaccharide (LPS)-binding protein (LBP) [5 ] is the focus of the present work. During myeloid differentiation into neutrophils, distinct classes of maturation-specific cytoplasmic granules are formed sequentially: primary (azurophilic), secondary (specific), and gelatinase granules [reviewed in ref. 6 ]. These are storage compartments for agents destined for release into the phagocytic vacuole or the extracellular space. The peroxidase-positive azurophil granules are formed at the promyelocytic stage of maturation and store antibiotic proteins such as BPI and defensins as well as digestive enzymes [7 ]. The proteins of azurophil granules generally undergo posttranslational modifications, including glycosylation and proteolytic trimming before or subsequent to storage [7 ]. A pro-region segment appears to be essential for the normal subcellular sorting of the defensins [8 ] and the propeptide of proMPO has a critical role in targeting for storage [9 , 10 ]. In contrast, deletion of the amino-terminal activation peptides and the carboxy-terminal prodomains is compatible with targeting for storage of the hematopoietic serine proteases [11 , 12 ]. At the least, a retention mechanism and efficient condensation are necessary for proteins destined for storage in myeloid granules so as to avoid constitutive secretion through a default pathway. However, individual sorting mechanisms may not be needed for each protein or type of granule because the timed expression of classes of proteins will keep them apart and allow separate storage [reviewed in ref. 6 ].

The specific bactericidal effect of BPI toward gram-negative bacteria is associated with a strong attraction for their outer envelope LPS [13 ]. This attraction explains in part the LPS-neutralizing activity of BPI [14 ]. The whole BPI molecule is not needed for the bactericidal and the LPS-neutralizing effects; a 25-kDa amino-terminal fragment promotes both activities [4 , 15 ], whereas the carboxy-terminal domain confers an opsonic function of holo-BPI [16 ] and has some LPS-neutralizing activity [4 ]. The carboxy-terminal half may also serve as an anchor in the membrane because of its prominent hydrophobicity [17 ] supported by the membrane-associated localization of BPI in the azurophil granules [18 ]. In addition, this location may protect against degradation, but either or both of the halves of BPI may be necessary for maintaining the stability of the molecule during processing, sorting, and storage. LBP, a circulating structural homolog of BPI, is normally produced by the liver [5 ] and exerts its function by binding to LPS and facilitating the delivery of LPS to CD14 on monocytes/macrophages [19 ]. In contrast, BPI neutralizes the biological effects of LPS. Despite the opposite responses to endotoxin, BPI and LBP share 44% sequence homology and both bind with their amino-terminal half to lipid A of LPS [20 21 22 ].

In this work we have investigated the role of the two domains of BPI in targeting for storage in granules. To this end, we expressed human BPI and LBP in the rat basophilic leukemia (RBL) [23 ] and the murine myeloid 32D [24 ] cell lines to investigate the processing and sorting for storage. These cell lines have previously been utilized for studies of the processing of neutrophil granule proteins [7 , 9 , 11 , 12 ]. Our aim was to examine the role of the amino-terminal and carboxy-terminal domains of BPI for protein stability and targeting for storage. Thus, we determined the consequences of deletion of the carboxy-terminal or the amino-terminal half, respectively, for posttranslational processing, targeting for storage, and stability in myeloid cells. Two chimeric proteins, BPI/LBP and LBP/BPI, that contain the amino- and the carboxy-terminal domain of BPI or LBP were also examined. Our results suggest a function of the carboxy-terminal half of BPI in the intracellular stability of the holoprotein and a potential role for the amino-terminal half of BPI in sorting.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
The eukaryotic expression vectors pRC/CMV and pcDNA3 were from Invitrogen (Groningen, The Netherlands). The vectors provide a cytomegalovirus promoter-driven expression of introduced cDNA. The plasmids also confer resistance to geneticin, allowing selection of transformants. (35S)Methionine/(35S)cysteine (cell labeling grade) and lactacystin were from ICN Pharmaceuticals (Costa Mesa, CA). TNT® Coupled Reticulocyte Lysate System was from Promega (Madison, WI). Percoll and protein A-Sepharose CL-4B were from Pharmacia (Uppsala, Sweden). Brefeldin A (BFA) and N-acetyl-leu-leu-norleucinal (ALLN) were from Sigma-Aldrich (St. Louis, MO). Geneticin and CompleteTM (protease inhibitor cocktail tablets) were from Boehringer Mannheim (Mannheim, Germany). Novex Pre-Cast Gels (10–20% Tris-Glycine Gel) were from Novex (San Diego, CA). Polyclonal antiserum to BPI was obtained by immunization of rabbits [1 ]. Rabbit polyclonal antiserum to LBP was a gift from XOMA (US) LLC (Berkeley, CA).

cDNA, mutagenesis, and construction of expression vectors
The human cDNA for BPI was generously provided by Dr. P. Elsbach, New York University, New York, NY. All 5’- and 3’-noncoding sequences were removed, the "Kozak" consensus leader sequence for maximal translational efficiency [25 ] was introduced, and the flanking restriction enzyme sites HindIII and NotI were added for subsequent cloning into plasmid. To this end polymerase chain reactions (PCR) were performed with full-length BPI cDNA as template in a 20-cycle reaction with upstream primer 5’-GACTTCAGAAGCTTCCGCCACCATGAGAGAGAACATGGCCAGGGGC and downstream primer 5’-GACTTCAGGCGGCCGCTCATTTATAGACAACGTCTGC (start and stop codons in boldface and restriction enzyme sites underlined). The resulting product was cloned into the expression vectors pcDNA3 and pMPSV-H for construction of the expression vectors pcDNA3-BPIwt and pMPSV-H-BPIwt. Control sequencing showed preserved integrity of the reading frame.

The amino-terminal half and three other carboxy-terminal deletion mutants of BPI were formed by PCR amplifications of BPI cDNA with the following downstream primers: 5’-GACTTCAGGCGGCCGCTCATGGAGGTGCCACCAGACCATAG for introduction of a stop codon (boldface) after Pro213 (numbered from the first amino acid of the mature protein), 5’-GACTTCAGGCGGCCGCTCACTCAGGTAGGAAGGTTCCAAAG for introduction of a stop codon after Glu304, 5’-GACTTCAGGCGGCCGCTCACAGGAGCAGCCTATCCAGC for introduction of a stop codon after Leu386, and 5’-GACTTCAGGCGGCCGCTCAGAGAGGGAAGCCTTTCTGTAG for introduction of a stop codon after Leu427. The deletion mutants were cloned into the pcDNA3 and pMPSV-H vectors to create the expression vectors pcDNA3-BPI1–213, -BPI1–304, -BPI1–386, and -BPI1–427, as well as pMPSV-H-BPI1–213, -BPI1–304, -BPI1–386, and -BPI1–427.

The carboxy-terminal region of BPI (BPI210–456) was made with full-length BPI cDNA as a template in a two-step "spliced overlap extension" PCR [26 ]. In the first reaction two separate amplifications produced two fragments of BPI, one amino-terminal fragment including the signal peptide, and one fragment with the carboxy-terminal part of the protein. In designing the primers, the "Kozak" consensus leader sequence as well as the flanking restriction enzyme sites HindIII and NotI were included. The PCR primers in the two amplifications were upstream 5’-GACTTCAGAAGCTTCCGCCACCATGAGAGAGAACATGGCCAGGGGC (primer 1) plus downstream 5’-CGTGGTTGCTGGAGGTGCCACGGCCGCTGTCACGGCGGTGC (primer 2), and upstream 5’-GTGGCACCTCCAGCAACCAC (primer 3) plus downstream 5’-GACTTCAGGCGGCCGCTCATTTATAGACAACGTCTGC (primer 4), respectively. The PCR products were isolated on agarose gel, mixed, and subjected to a second splicing PCR amplification with primers 1 and 4, thus creating BPI lacking the amino acids 1–209 (BPI210–456). The resulting PCR product was cloned into plasmid (pcDNA3) to create the expression vector pcDNA3-BPI210–456. All PCRs were performed in a Perkin-Elmer 480 Thermal Cycler using Pfu polymerase (Stratagene, La Jolla, CA) according to the manufacturer’s instructions.

The plasmids p4161(B-L), p4160(L-B), and pLBP encoding the fusion proteins BPI1–199/LBP198–456, LBP1–197/BPI200–456, and the protein LBP, respectively, were provided by XOMA (US) LLC [27 ]. The inserts were cut out from the plasmids with the restriction enzymes SalI and XhoI. The 5’-end produced by SalI was made blunt with the Klenow enzyme. The inserts were then ligated to pcDNA3 that had been cut with EcoRV (blunt end) and with XhoI, to create the expression vectors pcDNA3-BPI/LBP, -LBP/BPI, and -LBP.

The cDNA constructs were investigated by in vitro translation and were found to encode for proteins of the expected molecular size as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The cDNA constructs used in this work are shown in Figure 1 .



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  		Figure 1. Schematic view of the cDNA constructs used. The numbers indicate the first and last amino acid in the respective peptide, numbered from the first amino acid of the mature protein. SP, signal peptide.

 
Cell culture
The rat basophilic/mast cell line RBL-1 and the murine myeloblast-like cell line 32D cl3 cells were cultured as described [28 ]. Exponentially growing cells were used in all experiments.

Transfection
The RBL-1 cells were transfected with pcDNA3 constructs through the use of the Bio-Rad Electroporation Apparatus (Bio-Rad, Hercules, CA), with electrical settings of 960 µF and 260 V as described previously [29 ]. After electroporation, 2 mg/mL geneticin was added to select for recombinant clones containing the geneticin-resistant gene of pcDNA3. The eucaryotic expression vector pMPSV-H contains a promoter from myeloproliferative sarcoma virus but confers no selection marker for eucaryotic cells. Therefore, the 32D cl3 cells were cotransfected with a pMPSV-H construct and the pRC/CMV vector or transfected with pcDNA3 constructs with the same electrical settings as above, and selected with 1 mg/mL geneticin. Individual antibiotic-resistant cell clones were selected, expanded in suspension cultures, and screened for the expression of the transfected protein by biosynthetic radiolabeling. The 32D cell clones of BPI, LBP, BPI/LBP, and LBP/BPI (see Fig. 7B ) are, however, not-clonal antibiotic-resistant transfectants.



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  		Figure 7. Differences in processing of BPIwt, LBP, BPI/LBP, and LBP/BPI. RBL cells (A) and 32D cells (B) transfected with cDNA for BPIwt, human LBP, the chimeric BPI/LBP, or LBP/BPI were incubated with [35S]methionine/[35S]cysteine for 30 min, followed by chase of the label for up to 5 h. At depicted time points, 20 x 106 cells were removed, and after lysis, subjected to immunoprecipitation with anti-LBP (LBP, BPI/LBP) or anti-BPI (BPIwt, LBP/BPI). In addition, the incubation medium was also investigated at each chase time. Immunoprecipitates were analyzed as described in the legend to Figure 2 . The fluorograms were exposed 12, 11, 12, and 16 days, respectively (A), and 18, 18, 13, and 13 days, respectively (B).

 
Biosynthetic radiolabeling
Biosynthetic radiolabeling of newly synthesized proteins was carried out as described previously [29 ]. Cells were starved for 30 min in methionine/cysteine-free medium, followed by radiolabeling with [35S]methionine/[35S]cysteine (15–100 µCi/mL) for 30 min (pulse labeling), unless otherwise indicated. For chase of the radiolabel, the cells were resuspended in complete medium. At timed intervals, cells were withdrawn and lysed for immunoprecipitation or homogenized for subsequent subcellular fractionation.

Subcellular fractionation
Subcellular fractionation was performed as described [29 ]. Thus, the postnuclear cell homogenate was centrifuged in a Percoll density gradient, after which nine fractions were collected with all the cytosol in fraction 9. The distribution of lysosomes and Golgi elements in the gradient was determined by assaying ß-hexosaminidase and galactosyl transferase as described elsewhere [30 , 31 ]. The peak activities of ß-hexosaminidase and galactosyl transferase in subcellular fractions from RBL cells were localized in fractions 1–2 (containing the dense cytoplasmic organelles referred to in this work) and 5–8, respectively, and from the 32D cells in fractions 2 and 6, respectively [28 ].

Immunoprecipitation
Before immunoprecipitation, whole cells were solubilized in a lysis buffer consisting of 1 M NaCl, 50 mM Tris-HCl, pH 8.0, 0.5% Triton X-100 with added protease inhibitors (Complete), frozen and thawed three times, whereas culture medium or Percoll-containing subcellular fractions were solubilized in radioimmune precipitation buffer (RIPA) [29 ], also including protease inhibitors. Biosynthetically radiolabeled BPIwt, truncated forms of BPI and LBP/BPI were immunoprecipitated with polyclonal anti-BPI while BPI/LBP and LBP were precipitated with polyclonal anti-LBP, followed by electrophoretic analysis and fluorography as described before [29 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of wild-type BPI and BPI1–213 in RBL and 32D cells
To determine whether the carboxy-terminal domain of BPI is necessary for sorting and/or stability of BPI, a number of truncated BPI cDNA species were produced. Stable clones with wild-type BPI and truncated species of BPI were established by transfection of the RBL and 32D cell lines. Results are mainly reported for full-length BPI (BPIwt) and for BPI1–213 lacking the carboxy-terminal half. Endogenous BPI was not detectable in the cell lines with the antiserum used (data not shown).

BPIwt is visualized as a single 55-kDa band (Fig. 2 ). A minor decrease of the intracellular product is seen in 32D cells (Fig. 2B) but not in RBL cells (Fig. 2A) during a 3-h chase of the radiolabeled BPI. Thus RBL cells retain synthesized BPI more efficiently than 32D cells. This is at least in part due to minimal secretion in RBL cells, whereas 32D cells secrete a large amount of newly synthesized BPI. Similar differences were observed for other neutrophil granule proteins expressed in these cells [32 , 33 ].



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  		Figure 2. Differences in stability and secretion of BPIwt and BPI1–213. RBL cells (A) and 32D cells (B) transfected with cDNA for human BPIwt or BPI1–213 were incubated with [35S]methionine/[35S]cysteine for 30 min, followed by chase of the label for 1 and 3 h. At depicted time points, 40 x 106 cells were removed and, after lysis, subjected to immunoprecipitation with anti-BPI. In addition, BPI was also precipitated from the incubation medium at each time point. The immunoprecipitates were analyzed by SDS-PAGE in a 10–20% gradient gel followed by fluorography. The fluorograms were exposed for 6 weeks (BPIwt) and 15 days (BPI1–213) in panel A, and 3 days and 33 days, respectively, in panel B. The positions of BPIwt and BPI1–213 are indicated to the right with arrows.

 
If the carboxy terminal is necessary for sorting, BPI1–213 would, unlike intact BPI, not be targeted for storage in dense organelles. If, on the other hand, the carboxy-terminal domain plays a role for folding of nascent protein, BPI1–213 would be misfolded and retained in the endoplasmic reticulum (ER). Finally, instability as a result of lack of the carboxy-terminal half might lead to degradation during processing and targeting. BPI1–213 is rapidly degraded in RBL cells (Fig. 2A) , indicated by the disappearance of the radiolabel after 1 h. It is more stable in 32D cells (Fig. 2B) , still detectable after 3 h of chase of the radiolabel. However, lower-molecular-weight degradation forms of intracellular BPI1–213 are visible with time in 32D cells, whereas the disappearance in RBL cells is too rapid to allow detection of any degradation forms. Thus, BPI1–213 is highly unstable, indicating that the carboxy-terminal half of BPI is necessary for creating a stable conformation that resists proteolytic degradation in the intracellular environment. Like BPIwt, BPI1–213 is also secreted in an undegraded form from 32D cells but barely from RBL cells (Fig. 2) . The deletion mutants BPI1–386 and BPI1–427 displayed a higher stability than BPI1–213 when expressed in 32D cells (data not shown). These mutants, in contrast to BPI1–213 and BPI210–456 have a preserved central ß-sheet that may be necessary for stability (see Discussion).

To determine whether newly synthesized BPI1–213 is degraded in the ER as a consequence of misfolding, pulse-chase experiments were carried out in the presence of BFA. BFA induces a disassembly of the Golgi complex, thereby preventing ER-Golgi transport [34 , 35 ]. The degradation of BPI1–213 is clearly diminished in both RBL (Fig. 3A ) and 32D cells (Fig. 3B) in the presence of BFA, and the lower-molecular-weight forms of intracellular BPI1–213 are not visible, indicating that the degradation is inhibited. The secretion of BPI1–213 is abolished by BFA. These results indicate that the degradation of BPI1–213 does not occur to a major extent in the ER, but rather after that the intracellular pathway for storage is separated from that for constitutive secretion. To determine the role of lysosomal degradation for instability, the cells were incubated with chloroquine or NH4Cl, thus blocking lysosomal proteolysis by raising the pH. Results from pulse-chase experiments carried out in the presence of either agent showed diminished degradation of BPI1–213 during chase of the radiolabeled product in both cell lines (Fig. 3) . However, all degradation was not abolished because a lower-molecular-weight form was still produced, indicating a pH-insensitive step. The difference in stability of BPI1–213 in RBL and 32D cells may reflect a difference in the proteolytic equipment of the two cell lines. In any case, these results indicate that the instability of BPI1–213 is caused by lysosomal/granule degradation.



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  		Figure 3. The effect of BFA, NH4Cl, and chloroquine on the processing of BPI1–213. RBL cells (A) and 32D cells (B) transfected with cDNA for BPI1–213 were incubated with [35S]methionine/[35S]cysteine for 30 min, followed by chase of the label for up to 3 h (controls). Separate experiments were carried out with 5 µg/mL brefeldin A, 10 mmol/L NH4Cl or 1 µmol/L chloroquine, with the agents present during starvation (30 min), pulse-labeling, and chase of the label. At indicated time points, 40 x 106 cells were removed and, after lysis, subjected to immunoprecipitation with anti-BPI. In addition, BPI1–213 were also precipitated from the incubation at each time point. Immunoprecipitates were analyzed as described in legend to Figure 2 . The fluorograms were exposed for 28–32 days. The position of BPI1–213 is indicated to the right with arrows.

 
Wild-type BPI is targeted to dense cytoplasmic organelles
BPI is normally targeted for storage in azurophil granules formed in promyelocytes [3 ]. Possible targeting of BPI for storage in dense organelles was investigated in RBL and 32D cells using pulse-chase radiolabeling experiments followed by subcellular fractionation (Fig. 4 ). After 30 min of radiolabeling, most of the labeled BPIwt in both cell lines is in the light density fractions corresponding to ER and Golgi elements [32 ]. After 1 h of chase, most of the radiolabel in RBL cells (Fig. 4A) and a part of that in 32D cells (Fig. 4B) is located in the dense fractions, indicating transfer to dense organelles. At each time point, some radiolabeled protein is also found in fraction no. 9, e.g., cytosol, due to disruption of intracellular membrane-bound organelles during the homogenization procedure. Thus, the RBL and 32D cell lines can target BPI to dense cytoplasmic organelles.



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  		Figure 4. Targeting of BPIwt to dense organelles. RBL cells (A) and 32D cells (B) transfected with cDNA for human BPIwt were incubated with [35S]methionine/[35S]cysteine for 30 min followed by chase of the label for 1 and 3 h. At these time points, 100 x 106 cells were homogenized, after which subcellular fractionation of the postnuclear supernatant was performed by centrifugation in Percoll, with subsequent collection of nine subcellular fractions, fraction no. 9 containing all cytosol. The fractions were lysed and subjected to immunoprecipitation with anti-BPI. Immunoprecipitates were analyzed as described in the legend to Figure 2 . The fluorograms were exposed for 6 and 8 days, respectively. The position of BPIwt is indicated with an arrow to the right. Peak activities of ß-hexosaminidase and galactosyl transferase were localized in fractions 1–2 and 5–8, respectively, in RBL cells and in fractions 2 and 6, respectively, in 32D cl3 cells. Peak activities of ß-hexosaminidase and galactosyl transferase indicate the position of lysosomes and Golgi elements, respectively.

 
The amino-terminal half of BPI, BPI1–213, is targeted to dense organelles followed by degradation
BPI1–213 was found to be unstable in both RBL and 32D cells (Fig. 2) . Stabilization of BPI1–213 with BFA, NH4Cl, or chloroquine (Fig. 3) suggested that degradation takes place in a lysosomal compartment. To investigate this further, subcellular fractionation was performed after pulse/chase labeling. BPI1–213 is at least in part shown to be translocated to dense organelles of 32D cells (Fig. 5A ). Because the degradation of BPI1–213 is almost complete after 1 h of chase of the label in RBL cells (Fig. 2A) , the subcellular fractionation was performed after pulse/chase labeling in the presence of chloroquine, in order to stabilize the radiolabeled protein. The translocation is very rapid with a radiolabeled product visible in dense organelles already after 30 min of chase (Fig. 5B) . However, even in the presence of chloroquine the dense organelle product is unstable because it is barely visible after 1 h of chase. BPI1–213 appears in two sizes, a larger size dominating in the light fractions and a smaller size dominating in the dense fractions, suggesting degradation or processing before transfer.



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  		Figure 5. Targeting of the amino-terminal half of BPI, BPI1–213, to dense organelles. 32D cells (A) and RBL cells (B) transfected with BPI1–213 were incubated with [35S]methionine/[35S]cysteine for 1 h and 30 min, respectively, followed by chase of the label for 1 and 3 h or 30 and 60 min, respectively, as indicated. RBL cells, but not 32D cells, were preincubated, pulse-labeled, and chased in the presence of 1 µmol/L chloroquine. At depicted time points, 100 x 106 cells were withdrawn and subjected to homogenization and subcellular fractionation as discribed in the legend to Figure 4 . The fluorograms were exposed for 33 days. The position of BPI1–213 is indicated to the right with arrows.

 
The carboxy-terminal half of BPI, BPI210–456, is retained in the ER followed by degradation by the proteasome
The results described above reveal that the carboxy-terminal half (amino acids 214–456) of BPI is important for stability but not required for sorting to storage in dense cytoplasmic organelles. The stability and targeting of the carboxy-terminal half of BPI (amino acids 210–456) was also investigated. To achieve a correct cleavage of the signal peptide we chose the beginning of the mutant protein to be Val210 (numbered from the first amino acid of the mature protein) because the natural cleavage point in BPI for the signal peptidase is a valine, Val1. The two created halves of BPI (BPI1–213 and BPI210–456) overlap each other with four amino acids. The cDNA of BPI210–456 consists of the signal peptide joined to the amino acid sequence 210–456, thus lacking the amino acids 1–209.

BPI210–456 is highly unstable in both RBL and 32D cells (Fig. 6 ). BPI210–456 is unlikely to be transferred to dense cytoplasmic organelles because the instability of the mutant protein is unchanged in the presence of chloroquine, NH4Cl, or BFA in both the RBL (Fig. 6A , compare with the results for BPI1–213 in Fig. 3 ) and the 32D cell line (data not shown). Therefore, BPI210–456 seems to be retained in the ER and degraded, most likely in the proteasome. This conclusion is supported by the finding of diminished degradation of BPI210–456 by N-acetyl-leu-leu-norleucinal (ALLN) in RBL (Fig. 6A) and 32D cells (data not shown), a cysteine protease inhibitor that blocks the 20S proteasome and ER degradation at the same time [36 , 37 ]. Moreover, a highly specific inhibitor of the 20S proteasome, lactacystin [36 , 38 ], diminishes the degradation of BPI210–456 in both RBL and 32D cells (Fig. 6B) , indicating retention in ER and subsequent proteasome degradation of BPI210–456. Some secretion of BPI210–456 becomes visible in RBL cells when the degradation of the mutant protein is diminished by lactacystin.



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  		Figure 6. Effects of BFA, NH4Cl, chloroquine, ALLN, and lactacystin on the processing of BPI210–456. RBL cells (A) and also 32D cells (B), transfected with cDNA for BPI210–456 were incubated with [35S]methionine/[35S]cysteine for 30 min, followed by chase of the label for indicated time periods (controls). Separate experiments were carried out with 5 µg/mL brefeldin A, 10 mmol/L NH4Cl, 1 µmol/L chloroquine, 50 µmol/L ALLN, or 10 µmol/L lactacystin present during starvation, pulse-labeling, and chase of the label. At indicated time points, 20 x 106 cells were removed and, after lysis, subjected to immunoprecipitation with anti-BPI. In addition, BPI21-–456 was also precipitated from the incubation medium at each chase time point. Immunoprecipitates were analyzed as described in the legend to Figure 2 . The position of BPI210–456 is indicated with arrows. The fluorograms were exposed for 8–9 days (A) and 18 days (B).

 
Processing and targeting of LBP and the chimeric BPI/LBP and LBP/BPI
The results above suggest a role of the amino-terminal half of BPI in targeting for storage and a role of the carboxy-terminal domain of BPI for stability of the sorted product. To explore further the role of the amino- and carboxy-terminal domains of BPI in processing and sorting, we examined the fate of chimeras between BPI and its plasma homolog LBP. BPI/LBP consists of the amino-terminal half of BPI (BPI1–199) fused with the carboxy-terminal domain of LBP (LBP198–456), and LBP/BPI consists of the amino-terminal domain of LBP (LBP1–197) fused with the carboxy-terminal half of BPI (BPI200–456). Stable clones of LBP, BPI/LBP, and LBP/BPI were established in RBL (Fig. 7A ) and 32D (Fig. 7B) cells. As might be expected, BPI seems to be better adapted for retention and storage in myeloid cells because it appears to be more stable than LBP (Fig. 7) , a secretory protein. However, the non-myeloid LBP is still surprisingly stable, particularly in RBL cells (Fig. 7A) . LBP accumulates in the dense cytoplasmic fraction of RBL cells (Fig. 8A ). LBP of two sizes is visible, the newly synthesized LBP has a smaller size than the LBP of the storage compartment. By replacing the carboxy-terminal half of LBP with the corresponding half of BPI the stability of the resulting hybrid (LBP/BPI) is increased in both cell lines compared with LBP (Fig. 7) . Transfer to a storage compartment is indicated by accumulation of the hybrid protein in the dense organelle fraction of RBL cells (Fig. 8B) . Thus, the carboxy-terminal half of BPI conferred increased stability, further supporting the conclusion from experiments described above with truncated BPI forms that the carboxy-terminal domain of BPI has a special role for the stability of the holoprotein in the storage environment. This is also consistent with the finding that BPI/LBP has a lower stability than BPI in both cell lines (Fig. 7) , but also that this hybrid accumulates in the dense cytoplasmic fraction of RBL cells (Fig. 8C) . Thus, the data on processing of BPI/LBP mutants, summarized in Figure 9 , indicate that the carboxy-terminal half of BPI is important for stability while the amino-terminal half of BPI (and LBP) may confer efficient targeting for storage.



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  		Figure 8. Targeting of LBP, LBP/BPI, and BPI/LBP to dense cytoplasmic organelles. RBL cells transfected with cDNA for human LBP (A), LBP/BPI (B), or BPI/LBP (C) were incubated with [35S]methionine/[35S]cysteine for 30 min, followed by chase of the label for 1 and 3 h. At times indicated, 100 x 106 cells were homogenized, after which subcellular fractionation was performed, with subsequent collection of nine subcellular fractions, fraction no. 9 containing all cytosol. The fractions were lysed and subjected to immunoprecipitation with anti-LBP (LBP, BPI/LBP) or anti-BPI (LBP/BPI). Immunoprecipitates were analyzed as described in the legend to Figure 2 . The fluorograms were exposed for 14 days. The position of respective protein is indicated with an arrow to the right. Peak activities of ß-hexosaminidase, fractions 1–2, and galactosyl transferase, fractions 5–8, indicate the position of lysosomes and Golgi elements, respectively.

 


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  		Figure 9. Summary of the results with full-length and chimeric BPI and LBP. A comparison of the stability, sorting, and secretion of BPIwt, BPI1–213, BPI210–456, LBP, BPI/LBP, and LBP/BPI in the RBL and 32D cell lines. The degree of stability, the amount of protein transferred to dense organelles (sorting) and the constitutive secretion is depicted. ND, not determined.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The primary aim of this research was to define the structural requirements for the intracellular processing of BPI: a unique antibiotic protein of azurophil granules of neutrophils. The structural requirements for the functions of BPI have been reported [16 , 39 ]. A proteolytic fragment consisting of residues 1–199, isolated after exposure of BPI to elastase, has been shown to contain the determinants necessary for the binding of LPS and the microbicidal activity toward Gram-negative organisms [15 , 40 ]. The present investigation has shown that human BPI, as well as its homologous plasma protein LBP, can be expressed in rodent hematopoietic cell lines to study processing and sorting for storage. Both proteins were targeted for storage in dense cytoplasmic organelles of RBL cells. Truncated forms of BPI as well as chimeras between BPI and LBP were also expressed. The amino-terminal half of BPI was, like full-length BPI, targeted for storage in dense organelles of both cell lines, but rapidly degraded after storage. In contrast, the carboxy-terminal half of BPI, when expressed, was retained in the ER and degraded. Chimeras between the amino-terminal and the carboxy-terminal halves of BPI and LBP, respectively, were, like full-length BPI, targeted for storage in dense organelles, although LBP/BPI showed higher stability than LBP or BPI/LBP. Thus, in this context, the carboxy-terminal half of BPI seems to play a role in stabilizing BPI to allow for long-term storage in cytoplasmic organelles that may also contain lysosomal enzymes responsible for degradation.

Stored BPI is very strongly associated with the granules and the most extensive solubilization of BPI has been achieved by extraction of cells with sulfuric acid [1 , 40 ]. A comparison by Western blotting between BPI extracted from RBL and 32D cells by sulfuric acid and the lysis buffer used in the present work (0.5% Triton X-100, 1 M NaCl) showed that sulfuric acid indeed gave the most thorough extraction (data not shown). However, sulfuric acid extracts are not appropriate for our purpose because some BPI will be lost during the dialysis of the samples carried out to allow the immunoprecipitation to take place. The density gradient fractions cannot be extracted with sulfuric acid because of agglutination of the Percoll. Therefore, the results might underestimate to some extent total stored amount of BPI. But only a minor decrease, if any, occurred of radiolabeled BPI during pulse-chase experiments carried out in both cell lines (Figs. 2 and 7) . Therefore, the recovery of the label seems to be unchanged and we conclude that the analysis reflects all the radiolabeled BPI present during the course of the experiments.

The myeloid cell lines as models for processing and sorting
The rodent myeloid cell lines RBL [23 ] and 32D [24 , 41 ] have been much utilized for studies of processing and sorting of human neutrophil granule proteins after transfection of their corresponding cDNAs [reviewed in ref. 42 ]. The RBL cells have cytoplasmic granules that are rich in serine proteases similar to azurophil granules of human neutrophils. These cells may therefore be suitable for studies of processing and sorting of neutrophil granule proteins such as serine proteases and BPI. The 32D cells can differentiate toward neutrophils during incubation with granulocyte colony-stimulating factor [41 ]. The cytoplasmic granules of 32D cells have an abnormal, vacuole-like appearance but can still function as phagolysosomes [24 , 43 ]. Thus, these cell lines are convenient for studies of processing and sorting of proteins destined for storage in myeloid granules. One major difference between these cells is seen in the efficiency of protein retention from secretion; RBL cells are more efficient than 32D cells in retaining a protein as opposed to constitutive secretion. Because these rodent cell lines do not correspond entirely to normal myeloid progenitor cells, additional studies will eventually be needed in normal hematopoietic progenitor cells by use of viral transfer of genes encoding granule proteins and their variants. The present study does not allow the exact localization of BPI, LBP, truncated variants, and chimeras targeted for storage in dense cytoplasmic organelles, including the granules of RBL cells and the vacuole-like granules of 32D cells, which may be heterogeneous. In this respect too, results from studies in normal progenitor cells, where feasible, should focus on subcellular localization in azurophil and specific granules as well as other organelles.

Both halves of BPI are necessary for stability but the amino-terminal half alone can be sorted for storage
Unlike most soluble proteins, BPI has a unique elongated shape with a pseudo-twofold symmetry [44 ]. Thus, it is organized in an amino-terminal and one carboxy-terminal domain (which correspond in this study to BPI1–213 and BPI210–456, respectively) connected by a proline-rich linker of 21 amino acids. Both the amino- and the carboxy-terminal domains contain a barrel formed by two {alpha}-helices and an antiparallel ß-sheet. The two barrels are connected by a smaller central antiparallel ß-sheet formed from the end and the beginning of each domain [44 ]. The three-dimensional structure of BPI is likely to determine the stability, since neither the amino- or carboxy-terminal domains of BPI were stable when expressed in RBL or 32D cells. An important role for the central ß-sheet of BPI in stabilization of the molecule was suggested by the finding of increased stability of deletion variants of BPI retaining the ß-sheet, compared with BPI1–213, lacking it.

The amino-terminal half of BPI (BPI1–213) accumulated in the dense cytoplasmic organelles indicating that the carboxy-terminal half may not be required for targeting. However, in contrast to full-length BPI, the accumulated amino-terminal half is unstable after transfer to dense organelles, indicating a requirement for the carboxy-terminal half to protect against degradation. An alternative possibility is that BPI may normally be protected against degradation as a result of its peripheral localization within azurophil granules [18 ]. In contrast, BPI1–193 [40 ] as well as rBPI23 and rBPI21 [45 ] are produced routinely as stable entities in non-myeloid cells. The stability may be explained by constitutive secretion and absence of the proteases that are abundant in myeloid cells. The carboxy-terminal half of BPI is hydrophobic and might mediate an association with the membrane. Likewise, although the amino-terminal domain of BPI binds to LPS of gram-negative bacteria, the carboxy-terminal domain of holo-BPI promotes opsonophagocytosis of the bacteria by mediating binding to the surface of the phagocyte [16 ]. The carboxy-terminal domain of BPI alone (BPI210–456) does not appear to folded in a manner that allows release from the ER. Thus, this half of BPI was degraded by a proteasome-mediated activity indicated by the increased stability in the presence of lactacystin, a specific proteasome inhibitor. Therefore, it is not possible to determine whether BPI210–456 also would accumulate in dense organelles if it could escape from the ER.

Our data suggest that the myeloid-derived protein BPI is better adapted than the secretory protein LBP for sorting for storage in myeloid cells because it is more stable than LBP. Because of the close relationship between BPI and LBP, the two-domain organization of BPI provides a model also for LBP [46 ] and the homologous organization of BPI and LBP is apparently conserved between different species [47 ]. The results from studies of the processing and sorting of chimeras between BPI and LBP further emphasized that the amino-terminal and the carboxy-terminal halves may have separate functions (Fig. 9) . Both hybrids are, like full-length BPI and LBP, targeted for storage. However, LBP/BPI is more stable than full-length LBP (or BPI/LBP), indicating that the carboxy-terminal domain of BPI may be adapted to confer protection against degradation in the storage compartment. Moreover, the amino-terminal domain of BPI may confer targeting for storage because BPI/LBP displayed lower secretion than LBP or LBP/BPI in both cell lines.

Mechanisms for targeting for storage in myeloid cells
Neutrophil granules are produced by fusion of coated vesicles derived from the trans-Golgi network. Sorting of proteins may depend on their conformation and aggregation. Studies have indicated that a mature conformation is compatible with storage in dense cytoplasmic organelles of hematopoietic serine proteases [11 , 42 ]. In this case, removal of the amino-terminal activation peptide, the carboxy-terminal prodomain, or the glycosylation sites does not interfere with targeting in 32D and RBL cells. However, the elimination of the propeptide of proMPO blocks sorting for storage in 32D cells [9 ]. Thus, in this case, a propeptide seems to carry a sorting signal for targeting or affect the physical state of proMPO that normally allows targeting. Likewise, the propeptide of defensins seems necessary to facilitate targeting for storage in dense organelles of 32D cells [8 ]. Unlike most neutrophil granule proteins, BPI is not modified by proteolytic trimming during subcellular sorting. Our results suggest that the protein conformation may be a determinant for sorting to storage granules to avoid constitutive secretion. Obviously, the conformation of the amino-terminal half of BPI when expressed alone (BPI1–213) allows targeting for storage but not stability. This observation exemplifies that successful storage depends on both targeting and stability at the storage site. The stability of BPI during sorting and storage was found to require the carboxy-terminal domain. Thus, the hydrophobic carboxy-terminal domain of BPI plays a role in opsonization extracellularly and a role for prevention of autodigestion in the intracellular environment. The segregation of neutrophil proteins into various storage compartments (granules) is probably a requisite for coexistence of biologically active constituents and the timed expression of the genes for the granule proteins coordinates the composition of each granule compartment so as to promote the stability of the included proteins [reviewed in ref. 6 ]. Our data suggest that, in addition to timed gene expression, the conformation of BPI is also important for long-term storage in granules.

Cell-specific targeting for storage
Myeloid cells are specialized in sorting for storage in granule compartments and may have an ability to segregate even non-myeloid proteins into regulated secretion (e.g., to granules). We show here that not only BPI but also the normally secretory LBP was efficiently sorted for storage in a myeloid cell line. Therefore, this result suggests that non-myeloid proteins can indeed be targeted to a myeloid cell storage compartment, indicating that sorting for storage may be cell-specific. The targeting of LBP may, however, be explained by a conformation that is closely similar to that of BPI, thus allowing sorting in myeloid cells. Moreover, even if heterologously expressed non-myeloid proteins are sorted for storage in a granule compartment of myeloid cells, they will not necessarily be resistant against degradation during storage.

Finally, what are the implications of these results for the understanding of determinants for sorting in myeloid cells to avoid constitutive secretion? In particular, our findings illuminate functions of the BPI domains. BPI has two functionally distinct domains with separate roles extra- and intracellularly. We propose a critical stability role for the hydrophobic carboxy-terminal domain during sorting for storage and a potential role for the amino-terminal domain in sorting.


   ACKNOWLEDGEMENTS
 
This work was supported by the Swedish Cancer Foundation, the Swedish Medical Research Council (project no. 11546), the Swedish Childhood Cancer Foundation, the Alfred Österlund Foundation, the Crafoord Foundation, the Greta and Johan Kock Foundation, and Funds of Landskrona-Lund-Orup Sjukvrdsdistrikt. We thank Dr. Peter Elsbach for the cDNA of BPI and XOMA (US) LLC, for LBP, BPI/LBP, and LBP/BPI plasmids and the LBP antibody. We are grateful to Ann-Maj Persson for expert technical assistence.

Received February 24, 2000; revised May 9, 2000; accepted May 10, 2000.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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