Molecular Vision 2002; 8:1-9 <http://www.molvis.org/molvis/v8/a1/> Received 9 November 2001 | Accepted 7 January 2002 | Published 11 January 2002

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Differential interaction of molecular chaperones with procollagen I and type IV collagen in corneal endothelial cells

MinHee K. Ko,¹ EunDuck P. Kay^1,2
 
 

¹Doheny Eye Institute and the ²Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA

Correspondence to: EunDuck P. Kay, D.D.S., Ph.D., Doheny Eye Institute, 1450 San Pablo Street, DVRC 203, Los Angeles, CA, 91202; Phone: (323) 442-6625; FAX: (323) 442-6688; email: ekay@hsc.usc.edu

Abstract

Purpose: Procollagen I is synthesized and intracellularly degraded in corneal endothelial cells (CEC), whereas type IV and VIII collagens are secreted into Descemet's membrane. In our previous study, we demonstrated that procollagen I synthesized by CEC is improperly folded and that the molecule was largely colocalized with protein disulfide isomerase (PDI) within the endoplasmic reticulum (ER). In the present study, we further investigated whether the a-subunit of prolyl 4-hydroxylase (P4Ha) and glucose regulatory protein/immunoglobulin heavy chain binding protein (Grp78/BiP) were also involved in ER retention of procollagen I in CEC.

Methods: Immunocytochemical analysis was performed to determine the colocalization of procollagen I with molecular chaprones. Protein synthesis was measured by immunoblot analysis and the association between proteins was determined by coimmunoprecipitation followed by immunoblot analysis. mRNA was quantitated using RT-PCR.

Results: To study the interaction of procollagen I with certain molecular chaperones involved in the collagen biosynthetic pathway, we determined whether procollagen I colocalized with P4Ha and Grp78/Bip, and then compared this molecular chaperone colocalization with their association with type IV collagen. Procollagen I was colocalized with either P4Ha or Grp78/Bip to a much lesser degree than type IV collagen was colocalized with these same ER proteins. Colocalization between the molecular chaperones demonstrated that P4Ha and Grp78/Bip were largely colocalized in the peripheral region of the ER, whereas colocalization of P4Ha and PDI was mostly limited to a small region of the ER. When cells were treated with a,a'-dipyridyl, the inhibitor did not affect the colocalization profiles of collagens with the molecular chaperones. However, the inhibitor markedly increased colocalization of P4Ha and PDI, but it significantly decreased colocalization between P4Ha and Grp78/Bip. When synthesis of the molecular chaperones was compared between CEC and corneal stromal fibroblasts (CSF), more Grp78/Bip and PDI were produced by CEC than by CSF. On the other hand, expression of Hsp47 was lower in CEC than it was in CSF. Coimmunoprecipitation was used to compare the association of P4Ha or Grp78/Bip with collagens in CEC and CSF. The association of collagens (regardless of type) with P4Ha or Grp78/Bip was much higher in CEC than in CSF. When the association of collagen molecules with respective molecular chaperones was compared in CEC, the degree of association between Grp78/Bip and procollagen I was similar to that between the molecular chaperone and type IV collagen. On the other hand, the degree of association between P4Ha and type IV collagen was much higher than that between P4Ha and procollagen I.

Conclusions: These data suggest that procollagen I and type IV collagen may use different molecular chaperones in the ER, thus targeting their distinctive destinations.

Introduction

Corneal endothelium is a monolayer of endothelial cells that covers the posterior surface of the cornea. The physiologic collagen phenotypes in corneal endothelial cells (CEC) are collagen types IV and VIII, the basement membrane collagens [1-3]. Collagen expression in CEC is unique in that CEC synthesize procollagen I, which is then intracellularly degraded immediately after its synthesis [4,5]. Secretion of procollagen I into Descemet's membrane would have an adverse affect on corneal function, including maintenance of transparency and transmittance of light. Thus, although CEC erroneously produce this undesirable procollagen I, they then block its secretion through intracellular degradation. This final stage of gene regulation acts as an important quality control mechanism, regulating the levels of many undesired proteins by blocking the secretion of mutant or malfolded proteins. It has been well documented that from 10% to 30% of newly synthesized procollagens are degraded intracellularly by a process termed basal degradation [6,7]; the degradation is markedly increased in those cells that synthesize procollagens with structurally abnormal triple helical domains [8,9]. Yet, the molecular basis of such intracellular degradation has not been fully defined. Furthermore, there are no studies concerned with how a single cell that produces a number of different collagen types recognizes a specific collagen type from its secreted counterparts and specifically degrades it, as is uniquely observed in CEC, under physiologic conditions.

The secretion of procollagen molecules is first regulated by the biosynthetic events that take place within the endoplasmic reticulum (ER). These events include assembly of three individual proa chains, the hydroxylation of prolyl and lysyl residues and the formation of the triple helix [10,11]. Numerous studies further demonstrate that newly synthesized procollagens in the ER are associated with molecular chaperones, such as Hsp47, glucose regulatory protein 78/immunoglobulin binding protein (Grp78/Bip), Grp94, and protein disulfide isomerase (PDI) [12-15]. This association suggests that these molecular chaperones assist in the procollagen folding process. In the ER, Grp78/Bip is known to be involved in retention of procollagen I that is synthesized by cells from osteogenesis imperfecta patients with mutations in the proa1(I) chain carboxyl-terminal propeptide [15,16]. Prolyl 4-hydroxylase (P4H) is also reported to induce ER retention of procollagen, independent of the enzymatic activity [17]. In addition to its role as the b-subunit of P4H [18,19], PDI acts as a molecular chaperone during the assembly of procollagen [20]. In previous studies, we have also reported that procollagen I in CEC preferentially binds to PDI [21] and that the association of procollagen I with Hsp47 is maintained at a very low level in CEC [22], despite its well-known specific binding to any collagen, including type I [12,23,24]. Our previous studies to elucidate the molecular basis of specific degradation of procollagen I in CEC demonstrated that the newly synthesized procollagen I is not properly folded, as evidenced by its pepsin-susceptibility [21]. In vitro pulse chase experiments [5] showed that the malfolded molecules were completely degraded inside the cells within 2 h after synthesis. Type IV collagen, in contrast, is secreted and deposited into Descemet's membrane [1].

The present study was undertaken to examine whether specific molecular chaperones within the ER regulate how the cell recognizes and retains unassembled or malfolded collagens. We compared the ER proteins bound to procollagen I and type IV collagen. From these ER proteins, we chose the two subunits of P4H for their critical roles as molecular chaperones and their role in the hydroxylation of procollagen [18-20,25]. We also chose Grp78/Bip for its binding capacity to procollagen molecules and the a-subunit of P4H (P4Ha) [15,16,26]. We examined the subcellular localization of procollagen I and type IV collagen, their respective colocalization profiles with the molecular chaperones, and the in vivo association profiles between collagen and these ER proteins. In addition, we compared expression levels of the ER proteins in CEC to their level in corneal stromal fibroblasts (CSF), which secrete type I collagen into the extracellular matrix (ECM). We have demonstrated that individual collagens may have preferential association potentials with the respective molecular chaperones in CEC. Procollagen I is largely associated with PDI, whereas type IV collagen is colocalized with P4Ha and Grp78/Bip.

Methods

Cell cultures

Rabbit CEC were isolated and cultures were established as previously described [1]. Briefly, the Descemet's membrane-corneal endothelium complex was treated with 0.2% collagenase and 0.05% hyaluronidase (Worthington Biochemical, Lakewood, NJ) for 60 min at 37 °C. Cultured cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Irvine Scientific, Santa Ana, CA) and 50 mg/ml of gentamicin (DMEM-10) in a 5% CO₂ incubator. First passage CEC were used for all experiments. For subculture, confluent cultures were treated with 0.2% trypsin and 5 mM EDTA for 3 to 5 min. To establish CSF, the corneas composed of stroma only were cut into small pieces and digested with collagenase cocktail for 3 to 4 h at 37 °C. The cells were resuspended in DMEM-10 and maintained and subcultured as described above. Third or fourth passage CSF were used for the study. In some experiments, cells were treated with 0.3 mM a,a'-dipyridyl for 2 h to inhibit the hydroxylation of collagens.

Protein preparation and protein determination

Cell cultures (90% confluent) were washed three times with phosphate-buffered saline (PBS). The cells were scraped in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 50 mM EDTA, 0.5 mM gelatin, 1 mM phenylmethylsulfonyl fluoride, 1 mM N-ethylmaleimide, 1 mg/ml leupeptin, and 1 mg/ml aprotinin) and then sonicated on ice. Protein concentration of the resultant lysates was assessed with a Bradford protein assay system [27].

Crosslinking

Procedures used for protein crosslinking were previously reported [22]. Dithiobis succinimidyl propionate (DSP), a substance that has been shown to cross cell membranes, was selected for this study. Cells were treated with 0.2% trypsin, containing 5 mM EDTA and 0.1% collagenase (Worthington Biochemical), to ensure removal of extracellular collagen. Cells were then combined with DSP (stored at a concentration of 0.1 M in dimethyl sulfoxide) to a final concentration of 2 mM, vortexed and placed on ice for 30 min. Following the reaction, cells were rinsed with 2 mM glycine in PBS to block the DSP activity and then with PBS alone. The cells were lysed with lysis buffer on ice for 15 min.

SDS-polyacrylamide gel electrophoresis

The conditions of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were as described by Laemmli, using the discontinuous Tris-Glycine buffer system [28].

Immunoprecipitation and immunoblot analysis

Cell lysates (5 mg) were precipitated with either anti-type I collagen antibody (3 mg) or anti-type IV collagen antibody (3 mg) for 18 h at 4 °C. The antigen-antibody complex was then precipitated with 50 ml of protein G-Sepharose beads (Sigma, St. Louis, MO) for 2 h at 4 °C. The precipitated immune complexes were washed three times with lysis buffer. The proteins bound to the Sepharose beads were eluted with Laemmli sample buffer [28], containing dithiothreitol, boiled for 5 min and applied to a 10% SDS-polyacrylamide gel for electrophoresis. The proteins separated by SDS-PAGE were transferred to a 0.45 mm nitrocellulose membrane (Bio-Rad Lab, Hercules, CA) at 0.22 ampere for 10 h in a semidry transfer system (transfer buffer: 0.1 M CAPS, pH 11). Immunoblot analysis was performed as described previously [21,22], using a commercial ABC Vectastain kit (Vector Laboratories, Burlingame, CA). All washes and incubations were carried out at room temperature in TTBS (0.9% NaCl, 100 mM Tris-HCl, pH 7.5, 0.1% Tween 20). Briefly, the nitrocellulose membrane was immediately placed in blocking buffer (10% nonfat milk in TTBS) for 1 h or left overnight at 4 °C. The membrane was then incubated with primary antibody (1:15,000 dilution for PDI; 1:10,000 for P4Ha, and 1:1000 dilution for Grp78/Bip) for 1 h and with biotinylated secondary antibody (1:5000 dilution) for 1 h. Incubation with ABC reagent was for 30 min. Membrane was treated with enhanced chemiluminescence (ECL) reagent (Amersham Pharmacia Biotech, Piscataway, NJ) and exposed to ECL film. The relative density of the polypeptide bands detected on ECL film was estimated using a one-dimensional image analyzer (LKB Ultrascan XL; Pharmacia LKB Biotechnology, Pleasant Hill, CA).

Relative quantitative reverse polymerase chain reaction

Total cellular RNAs from CEC and CSF were isolated using a QIAGEN RNeasy Mini kit (QIAGEN Inc, Valencia, CA), as described in the protocol provided with the kit. Relative quantitative reverse polymerase chain reaction (RQ RT-PCR) was also performed using QuantumRNA^TM Universal 18S Internal Standard (Ambion, Austin, TX), as described in the protocol provided by the manufacturer. Total RNA (2 mg), 5 mmole random primers for 18S internal standard [29], and 40 mM dNTP in nuclease-free water were heated to 85 °C for 3 min, then cooled on ice for 1 min. cDNA synthesis was initiated using 100 U of recombinant Moloney-murine leukemia virus reverse transcriptase at 42 °C for 60 min. The Grp78-specific primers were made from the cDNA sequences of human, rat, and hamster [30,31] to obtain the gene-specific primers to react with rabbit species. The sense primer was 5'-GACATCAAGTTCTTGCCGTT-3' and the antisense primer was 5'-CTCATAACATTTAGGCCAGC-3'. Before performing RQ RT-PCR, we determined the linear range reaction for Grp78/BiP in order to have the maximum amplification efficiency for both Grp78/Bip and the 18S internal standard. PCR was performed using a RoboCycler (Stratagene, La Jolla, CA). RQ RT-PCR was performed as follows; hot start for 5 min at 95 °C, followed by denaturation for 1 min at 95 °C, annealing for 1 min at 56 °C, and elongation for 1 min at 72 °C for a total of 28 cycles with 18S internal standards and competimers (4:6). The reaction mixtures were separated on a 2% agarose gel and stained with ethidium bromide to analyze the RQ RT-PCR product.

Immunofluorescent staining

CEC (3 x 10⁴/chamber) were seeded on 4-well chamber slides and maintained in culture until they reached 50% confluence. Cells were washed with PBS and fixed for 10 min with ice-cold methanol or 1% acetic acid in 95% ethanol, depending on the primary antibodies used. Cells were permeabilized and blocked with 0.1% Triton X-100 and 1% BSA in PBS for 15 min at room temperature. After washing, cells were simultaneously incubated with both primary antibodies prepared in PBS. The cells were incubated overnight at 4 °C or 1 h at room temperature and then washed with PBS. Cells were then simultaneously incubated with FITC-conjugated secondary antibody (1:100 dilution) and rhodamine-conjugated secondary antibody (1:200 dilution) for 30 min at 37 °C in the dark. After extensive washing with PBS, the slides were mounted in a drop of Vectashield mounting medium (Vector Laboratories) to reduce photobleaching. Control experiments were performed in parallel with the omission of one of the secondary antibodies. For double-staining experiments in which both primary antibodies were produced in the same species (mouse), the previously reported experimental procedures were used with a slight modification [21]. Cells were blocked with 5% normal goat serum after fixing and then incubated with the first primary monoclonal antibody at 37 °C for 1 h. Cells were then rinsed in PBS and incubated at 37 °C for 1 h with an excess of the unconjugated rabbit Fab antibody directed against mouse IgG (20 mg/ml dilution, Jackson Immunoresearch Laboratories, West Grove, PA). After extensive washing, cells were incubated with rhodamine-conjugated goat anti-rabbit IgG antibody (1:200 dilution) at 37 °C for 30 min in the dark. Following extensive washing in PBS, the second monoclonal antibody was incubated at 37 °C for 1 h and cells were rinsed in PBS. The corresponding secondary FITC-conjugated anti-mouse IgG (1:100 dilution) was incubated for 30 min at room temperature in the dark. Control experiments were performed in parallel with the omission of one of the primary antibodies. The staining profiles of the control experiments were examined exclusively and the data were not shown in the text.

Confocal microscopy and image analysis

Antibody labeling was examined using a Zeiss LSM-510 laser scanning confocal microscope. Optical slices (1.8 mM) were taken perpendicular to the cell monolayer (apical to basal orientation). A 488-nm Argon laser was used in combination with a 499/505-530 excitation/emission filter set for fluorescein examination. For rhodamine, the 543-nm Helium neon laser was used with a 543 excitation filter and 560 emission filter. Simultaneous images of FITC or rhodamine were captured from the same optical section. The captured images were then pseudocolored: red for rhodamine and green for FITC. Regions of colocalization appear in yellow, reflecting the additive effect of superimposing green and red pixels. Image analysis was performed using the standard system operating software provided with the Zeiss LSM-510 series microscope.

Antibodies

Mouse anti-P4Ha antibody was purchased from ICN. Mouse monoclonal anti-PDI antibody and rabbit anti-Grp78 polyclonal antibody for immunoblotting analysis were purchased from Stressgen Biotechnologies Corp. (Victoria, BC, Canada), while mouse anti-Grp78 monoclonal antibody for immunofluorescence staining was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse anti-type IV collagen antibody was a gift from Dr. Nirmala SundarRaj (University of Pittsburgh, Pittsburgh, PA), and goat anti-type I collagen was purchased from Chemicon International, Inc. (Temecula, CA). FITC-conjugated goat antibody against mouse IgG, FITC-conjugated rabbit antibody against goat IgG, rhodamine-conjugated horse antibody against mouse IgG, and rhodamine-conjugated rabbit antibody against goat IgG were purchased from Vector Laboratories Inc. Goat serum, Fab fragment anti-mouse IgG (H+L), and rhodamine-conjugated goat anti-rabbit IgG were purchased from Jackson Immunoresearch Laboratories. Primary antibodies were used for immunofluorescent staining in the following dilutions: anti-type I collagen (1:50); anti-type IV collagen (1:200); anti-P4Ha (1:200); anti-PDI (1:200); anti-Grp78/Bip (1:400).

Results

Subcellular localization

In the previous study, we examined the subcellular localization of P4H containing both subunits, and PDI (the b subunit). The subcellular localization of the a-subunit of P4H was not determined. Therefore, for the present study, we examined the subcellular localization of P4Ha along with PDI and of Grp78/Bip, the key molecular chaperones during procollagen biosynthetic events. Figure 1 shows an even distribution of P4Ha throughout the ER (Figure 1A) and a diffuse distribution of PDI, with some areas lacking the protein (Figure 1B). Distribution of Grp78/Bip was largely localized toward the peripheral region of the ER, while there was much less perinuclear staining (Figure 1C). When CEC were double-stained with antibodies against type I collagen and P4Ha, a minor fraction of the two proteins was colocalized at the perinuclear ER, however, the major portion of the two proteins was not colocalized (Figure 2A). In contrast, type IV collagen and P4Ha were largely codistributed throughout the ER (Figure 2B). When cells were treated with a,a'-dipyridyl, an ion chelator that inhibits hydroxylation of prolyl residues in procollagen chains, colocalization profiles of procollagen I and P4Ha was not altered from those of the untreated cells (Figure 2C). On the other hand, the unhydroxylated type IV collagen showed a large degree of colocalization with P4Ha throughout the ER (Figure 2D), similar to that observed in untreated cells.

Grp78/BiP is known to stably bind malfolded procollagen I that has mutation in the carboxy-terminal propeptide in fibroblasts from patients with osteogenesis imperfecta [15,16]. We examined whether procollagen I synthesized in CEC was associated with Grp78/Bip. When cells were double-stained with antibodies against type I collagen and Grp78/Bip, the colocalization of the two proteins was shown in the restricted areas of the ER if only Grp78/Bip was present (Figure 3A,C). Type IV collagen and Grp78/BiP were largely colocalized at the peripheral region of the ER, where the molecular chaperone was predominantly present (Figure 3E,G). Of interest is that procollagen I was distributed throughout the ER (Figure 3B), whereas a more restricted distribution of type IV collagen was observed in the ER of CEC (Figure 3F). When collagen molecules were unhydroxylated with the treatment of a,a'-dipyridyl, the colocalization profile of procollagen I and Grp78/Bip was unaltered compared to that of the untreated cells (Figure 3D). However, a,a'-dipyridyl promotes the colocalization of type IV collagen and Grp78/Bip throughout the ER and even at perinuclear region of the ER (Figure 3H).

The unassembled a-subunits of P4H are associated with Grp78/Bip, leading to an assembly-competent form in the ER [26]. Therefore, we determined whether P4Ha in CEC was colocalized with Grp78/Bip. When cells were double-stained with antibodies against P4Ha and Grp78/BiP, the two proteins were largely colocalized in the peripheral region of the ER (Figure 4A). But when PDI and P4Ha were analyzed for codistribution, the two subunits of the P4H enzyme were not largely colocalized (Figure 4B). Under the stress condition mediated by a,a'-dipyridyl, the colocalization profile of P4Ha and Grp78/Bip in the treated cells (Figure 4C) was less than that in the untreated cells (Figure 4A). On the other hand, P4Ha and PDI showed complete colocalization in the cells treated with a,a'-dipyridyl (Figure 4D), suggesting that the two subunits of the enzyme were preferentially associated with each other when underhydroxylated procollagen molecules prevailed.

Expression and association of chaperones with collagen

Our previous study demonstrated that the expression level of Hsp47 in CEC was much less than that of CSF at both the protein and mRNA levels [22], whereas CEC contained much higher amounts of PDI than did CSF [21]. Since these measurements were performed separately, we determined the expression level of PDI, Hsp47, and Grp78/Bip simultaneously. The relative expression of these three molecular chaperones in CEC was compared to that of the CSF that served as control cells secreting type I collagen into the ECM. Three different concentrations were used to semi-quantitatively determine the expression levels of the ER proteins. The amount of Grp78/BiP and PDI in CEC were higher than the corresponding amounts in CSF, but the amount of Hsp47 was much lower in CEC (Figure 5). When the expression levels of these proteins were compared within the same cells, the levels of PDI and Hsp47 in CEC were similar, but the expression level of Grp78/Bip was low. In CSF, on the other hand, the expression level of Hsp47 was much greater than those of PDI and Grp78/Bip. When the relative density of the respective peptide bands was estimated, there was an approximate 50% increase in Grp78/BiP levels, an approximate two-fold increase in PDI, and a 25% decrease in Hsp47 in CEC compared to that in CSF (Figure 5). The steady state level of Grp78/BiP mRNA in CEC was examined using RQ RT-PCR analysis (Figure 6). The primers used for the analysis of Grp78/Bip generated a product of 260 bp, whereas the 18S rRNA used as the endogenous control generated a product of 310 bp. The steady state level of Grp78/BiP mRNA was much higher in CEC than in CSF, while the levels of 18S internal standard were similar in both cells.

We then investigated the association of procollagen I and type IV collagen with the molecular chaperones. The membrane permeable homobifunctional cleavable cross-linking reagent DSP was used before cell lysis to probe the in vivo association of chaperones with collagen molecules. A fraction of the cell lysates was immunoprecipitated with collagen antibodies, then immunoblotted with anti-Grp78 antibody or anti-P4Ha antibody (Figure 7). The association profiles between collagen molecules with molecular chaperones in CEC were compared with those in CSF. The immune complexes precipitated with either anti-type I collagen antibody or anti-type IV collagen antibody showed that the association of Grp78/Bip with collagen molecules, regardless of collagen types, was far greater in CEC than in CSF. A similar finding was observed in the association of collagen molecules with P4Ha; more collagen molecules were associated with P4Ha in CEC than in CSF. When the association of collagen molecules with respective molecular chaperones was compared in CEC, the degree of association between Grp78/Bip and procollagen I was similar to that between the molecular chaperone and type IV collagen. On the other hand, the degree of association between P4Ha and type IV collagen was much higher than that between P4Ha and procollagen I. This confirms the finding that colocalization of type IV collagen with P4Ha was much greater than the colocalization of procollagen I and P4Ha (Figure 2A,B).

Discussion

The study reported here grew out of an attempt to investigate the expression and subsequent intracellular degradation of procollagen I in CEC. CEC synthesize not only types IV and VIII collagen, but procollagen I as well. Upon secretion, types IV and VIII collagen are deposited into the network-forming basement membrane [1-3], whereas procollagen I is intracellularly degraded, as evidenced by in vitro pulse-chase experiments and in vivo staining of the corneal tissues [5]. Selective intracellular degradation is the major quality control mechanism preventing the secretion of unassembled and improperly folded proteins [11,32,33]. Our previous studies of the intracellular degradation of procollagen I in CEC demonstrate that intracellular procollagen I with a correct composition of two proa1(I) and one proa2(I) chains is pepsin-sensitive. This suggests that the molecule has an unstable triple helical conformation [21,22]. We also showed that this improperly folded procollagen I is preferentially bound to PDI (the b subunit of P4H) throughout the ER. Since CEC routinely operate the post-translational modification enzyme systems for types IV and VIII collagen, procollagen I can also be an equally specific substrate to the modification enzyme P4H. However, our previous study shows that procollagen I may not use such a unique modification step for procollagen biosynthetic events; instead, procollagen I preferentially binds to PDI. This extensive association with PDI may prohibit the binding of the molecule to the active P4H enzyme complex.

In the present study, we further examined differences in the behavior of procollagen I from that of type IV collagen in the ER before they follow their respective destined pathways; one is targeted to the cytosolic degradation site, the other is guided to the normal secretory pathway via the Golgi apparatus. From the ER resident proteins, we chose two subunits of P4H for their critical role in hydroxylation of procollagen chains and their additional roles as molecular chaperones, and we chose Grp78/Bip for its binding capacity to procollagen molecules and the a-subunit of P4H. Our data demonstrate that, under normal conditions, P4Ha appears to be associated with Grp78/Bip more than it binds with its b-subunit (PDI) in CEC. This finding is in agreement with the previous report in which the unassembled P4Ha forms complexes with Grp78/Bip until the P4Ha possesses an adequate secondary structure to prevent aggregation [26]. Note that under the stress condition induced by treating the cells with a,a'-dipyridyl, P4Ha is readily dissociated from Grp78/Bip and makes active enzyme complexes with PDI. It is interesting that the stress condition facilitates the disassociation of P4Ha from Grp78/Bip and promotes formation of the active enzyme complex between the two subunits. However, the underlying mechanism for this event is not known.

Our data further demonstrate that a minor fraction of procollagen I was colocalized with P4Ha at the perinuclear region of the ER and that the majority of the two proteins were not colocalized. On the other hand, subcellular localization of type IV collagen and P4Ha is largely coincidental throughout the ER. Placing the cells under stress conditions by treating them with a,a'-dipyridyl does not alter the unique subcellular localization of procollagen I and type IV collagen with P4Ha. These data suggest that neither procollagen I nor type IV collagen associated with P4Ha is hydroxylated under physiological conditions. This is consistent with the fact that the substrate of P4H is unhydroxylated collagen molecules. Colocalization of collagen molecules with Grp78/Bip also demonstrates that procollagen I shows much less colocalization with this ER resident protein than type IV collagen does with Grp78/Bip. Interestingly, the degree of association of procollagen I with Grp78/Bip at the protein level is similar to that between the molecular chaperone and type IV collagen. This observation, however, does not influence our interpretation of the immunohistochemical analysis because CEC contain much less Grp78/Bip than they do PDI or Hsp47 (Figure 5). The present findings and our previous data taken together show that procollagen I is largely colocalized with PDI in the ER rather than with P4Ha, Grp78/Bip, or Hsp47. On the other hand, type IV collagen is largely colocalized with P4Ha and Grp78/Bip. These data thus suggest that individual collagens may have preferential association potentials with their respective molecular chaperones. It is likely that the extensive binding of PDI to procollagen I inhibits the association of procollagen I with its active enzyme complex, P4H, thus leading to unhydroxylated molecules. Unhydroxylated procollagen I subsequently forms an unstable triple helix, resulting in the improperly folded molecule. Finally, this improperly folded procollagen I is recognized as a bad quality protein by the quality control system operating in the ER. This scenario may be further supported by the in vivo binding data which shows that P4Ha binds much less to procollagen I than to type IV in CEC. On the other hand, type IV collagen in CEC binds equally well to both subunits of P4H, thus, it is properly hydroxylated and folded. As a consequence, type IV collagen is transported out of the ER to the Golgi apparatus and secreted into the ECM. A recent study demonstrated that the C-propeptide from the proa2(I) chain is retained within the cell, where it forms a complex with PDI [34]. Although this study suggests that PDI binds to C-propeptide of proa chains, it is unknown whether PDI is associated with procollagen I via the individual C-propeptide chain of proa1(I) or proa2(I), or the trimeric C-propeptide in CEC. Furthermore, whether PDI interacts with the (Gly-X-Y) repeat domain as well as with the C-propeptide domain is yet to be determined.

Another interesting finding from the present study and our previous study [21] is the differential amounts of respective molecular chaperones in the respective cell lines. CEC produce more PDI, P4Ha, and Grp78/Bip than do CSF. On the other hand, CEC produce less Hsp47 than do CSF. The excess amount of PDI in CEC may be required for the ER retention of procollagen I, whereas the higher level of Grp78/Bip in CEC than in CSF may be caused by the presence of unfolded procollagen I, as reported in other systems [35]. It is also likely that CEC, containing less but sufficient Hsp47, need this particular molecular chaperone for their physiologic collagens, type IV and VIII. This is supported by the report that Hsp47 interacts with and stabilizes correctly folded procollagens [36]. In CEC, types IV and VIII collagen are the binding partners of Hsp47, as they meet the requirement of the molecular chaperone. Our previous report that procollagen I and Hsp47 have much less in vivo association in CEC than in CSF [22] further suggests that procollagen I may not be correctly folded in CEC.

The data presented in this study may suggest that procollagen I, preferentially bound to PDI, is directed to the degradation site, whereas type IV collagen, bound to the normal molecular chaperones and the modifying enzymes, is guided to complete the full pathway for secretion. It is not known whether the excessive binding of PDI to procollagen I causes improper folding of procollagen I and the subsequent ER retention, or whether the improperly folded procollagen I is protected by the prolonged association with PDI before the molecule is targeted to the degradation site. Although several cellular compartments have been identified as sites for degradation, including the lysosomes, a post-Golgi non-lysosomal compartment, the ER, and the proteasome system [37-39], it is unknown precisely where procollagen I is degraded in CEC.

Acknowledgements

Support for this work was provided by NIH grants EY06431 and EY03040, and Research to Prevent Blindness (New York, NY).

References

1. Kay EP, Smith RE, Nimni ME. Basement membrane collagen synthesis by rabbit corneal endothelial cells in culture. Evidence for an alpha chain derived from a larger biosynthetic precursor. J Biol Chem 1982; 257:7116-21.

2. Sawada H, Konomi H, Hirosawa K. Characterization of the collagen in the hexagonal lattice of Descemet's membrane: its relation to type VIII collagen. J Cell Biol 1990; 110:219-27.

3. Yamaguchi N, Benya PD, van der Rest M, Ninomiya Y. The cloning and sequencing of alpha 1(VIII) collagen cDNAs demonstrate that type VIII collagen is a short chain collagen and contains triple-helical and carboxyl-terminal non-triple-helical domains similar to those of type X collagen. J Biol Chem 1989; 264:16022-9.

4. Kay EP, He YG. Post-transcriptional and transcriptional control of collagen gene expression in normal and modulated rabbit corneal endothelial cells. Invest Ophthalmol Vis Sci 1991; 32:1821-7.

5. Kay EP, Gu X, Choi SH, Ninomiya Y. Posttranslational regulation of type I collagen in corneal endothelial cells. Invest Ophthalmol Vis Sci 1996; 37:11-9.

6. Bienkowski RS, Curran SF, Berg RA. Kinetics of intracellular degradation of newly synthesized collagen. Biochemistry 1986; 25:2455-9.

7. Neblock DS, Berg RA. Decreased synthesis and increased intracellular degradation of newly synthesized collagen in freshly isolated chick tendon cells incubated with monensin. Biochemistry 1986; 25:6208-13.

8. Bateman JF, Mascara T, Chan D, Cole WG. Abnormal type I collagen metabolism by cultured fibroblasts in lethal perinatal osteogenesis imperfecta. Biochem J 1984; 217:103-15.

9. Berg RA, Schwartz ML, Rome LH, Crystal RG. Lysosomal function in the degradation of defective collagen in cultured lung fibroblasts. Biochemistry 1984; 23:2134-8.

10. McLaughlin SH, Bulleid NJ. Molecular recognition in procollagen chain assembly. Matrix Biol 1998; 16:369-77.

11. Lamande SR, Bateman JF. Procollagen folding and assembly: the role of endoplasmic reticulum enzymes and molecular chaperones. Semin Cell Dev Biol 1999; 10:455-64.

12. Nagata K. Hsp47: a collagen-specific molecular chaperone. Trends Biochem Sci 1996; 21:22-6.

13. Hosokawa N, Nagata K. Procollagen binds to both prolyl 4-hydroxylase/protein disulfide isomerase and Hsp47 within the endoplasmic reticulum in the absence of ascorbate. FEBS Lett 2000; 466:19-25.

14. Ferreira LR, Norris K, Smith T, Hebert C, Sauk JJ. Association of Hsp47, Grp78, and Grp94 with procollagen supports the successive or coupled action of molecular chaperones. J Cell Biochem 1994; 56:518-26.

15. Chessler SD, Byers PH. BiP binds type I procollagen pro alpha chains with mutations in the carboxyl-terminal propeptide synthesized by cells from patients from osteogenesis imperfecta. J Biol Chem 1993; 268:18226-33.

16. Lamande SR, Chessler SD, Golub SB, Byers PH, Chan D, Cole WG, Sillence DO, Bateman JF. Endoplasmic reticulum-mediated quality control of type I collagen production by cells from osteogenesis imperfecta patients with mutations in the pro alpha 1 (I) chain carboxyl-terminal propeptide which impair subunit assembly. J Biol Chem 1995; 270:8642-9.

17. Walmsley AR, Batten MR, Lad U, Bulleid NJ. Intracellular retention of procollagen within the endoplasmic reticulum is mediated by prolyl 4-hydroxylase. J Biol Chem 1999; 274:14884-92.

18. Kivirikko KI, Myllyla R, Pihlajaniemi T. Protein hydroxylation: prolyl 4-hydroxylase, an enzyme with four cosubstrates and a multifunctional subunit. FASEB J 1989; 3:1609-17.

19. Kivirikko KI, Myllyharju J, Pihlajaniemi T. Hydroxylation of proline and lysine residues in collagens and other animal and plantproteins. In: Harding JJ, Crabbe MJC, editors. Post-translational modifications of proteins. Boca Raton: CRC Press; 1992. p. 1-51.

20. Wilson R, Lees JF, Bulleid NJ. Protein disulfide isomerase acts as a molecular chaperone during the assembly of procollagen. J Biol Chem 1998; 273:9637-43.

21. Ko MK, Kay EP. Subcellular localization of procollagen I and prolyl 4-hydroxylase in corneal endothelial cells. Exp Cell Res 2001; 264:363-71.

22. Gu X, Ko MK, Kay EP. Intracellular interaction of Hsp47 and type I collagen in corneal endothelial cells. Invest Ophthalmol Vis Sci 1999; 40:289-95.

23. Sauk JJ, Smith T, Norris K, Ferreira L. Hsp47 and the translation-translocation machinery cooperate in the production of alpha 1(I) chains of type I procollagen. J Biol Chem 1994; 269:3941-6.

24. Satoh M, Hirayoshi K, Yokota S, Hosokawa N, Nagata K. Intracellular interaction of collagen-specific stress protein HSP47 with newly synthesized procollagen. J Cell Biol 1996; 133:469-83.

25. John DC, Grant ME, Bulleid NJ. Cell-free synthesis and assembly of prolyl 4-hydroxylase: the role of the beta-subunit (PDI) in preventing misfolding and aggregation of the alpha-subunit. EMBO J 1993; 12:1587-95.

26. John DC, Bulleid NJ. Intracellular dissociation and reassembly of prolyl 4-hydroxylase: the alpha-subunits associated with the immunoglobulin-heavy-chain binding protein (BiP) allowing reassembly with the beta-subunit. Biochem J 1996; 317:659-65.

27. Kay EP, Lee MS, Seong GJ, Lee YG. TGF-beta s stimulate cell proliferation via an autocrine production of FGF-2 in corneal stromal fibroblasts. Curr Eye Res 1998; 17:286-93.

28. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680-5.

29. Pola R, Ling LE, Silver M, Corbley MJ, Kearney M, Blake Pepinsky R, Shapiro R, Taylor FR, Baker DP, Asahara T, Isner JM. The morphogen Sonic hedgehog is an indirect angiogenic agent upregulating two families of angiogenic growth factors. Nat Med 2001; 7:706-11.

30. Haas IG, Meo T. cDNA cloning of the immunoglobulin heavy chain binding protein. Proc Natl Acad Sci U S A 1988; 85:2250-4.

31. Normington K, Kohno K, Kozutsumi Y, Gething MJ, Sambrook J. S. cerevisiae encodes an essential protein homogolous in sequence and function to mammalian BiP. Cell 1989; 57:1223-36.

32. Klausner RD, Sitia R. Protein degradation in the endoplasmic reticulum. Cell 1990; 62:611-4.

33. Jentsch S, Schlenker S. Selective protein degradation: a journery's end within the proteasome. Cell 1995; 82:881-4.

34. Bottomley MJ, Batten MR, Lumb RA, Bulleid NJ. Quality control in the endoplasmic reticulum: PDI mediates the ER retention of unassembled procollagen C-propeptides. Curr Biol 2001; 11:1114-8.

35. Morris JA, Dorner AJ, Edwards CA, Hendershot LM, Kaufman RJ. Immunoglobulin binding protein (BiP) function is required to protect cells from endoplasmic reticulum stress but is not required for the secretion of selective proteins. J Biol Chem 1997; 272:4327-34.

36. Tasab M, Batten MR, Bulleid NJ. Hsp47: a molecular chaperone that interacts with and stabilizes correctly-folded procollagen. EMBO J 2000; 19:2204-11.

37. Ripley CR, Bienkowski RS. Localization of procollagen I in the lysosome/endosome system of human fibroblasts. Exp Cell Res 1997; 236:147-54.

38. Fitzgerald J, Lamande SR, Bateman JF. Proteasomal degradation of unassembled mutant type I collagen pro-alpha 1(I) chains. J Biol Chem 1999; 274:27392-8.

39. Mori K. Tripartite management of unfolded proteins in the endoplasmic reticulum. Cell 2000; 101:451-4.