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(Investigative Ophthalmology and Visual Science. 2000;41:4232-4239.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

Influence of Specific Regions in Lp82 Calpain on Protein Stability, Activity, and Localization within Lens

Hong Ma1, Marjorie Shih1, Chiho Fukiage1,2, Mitsuyoshi Azuma1,2, Melinda K. Duncan3, Nathan A. Reed3, Isabelle Richard4, Jacques S. Beckmann5 and Thomas R. Shearer1

1 From the Departments of Oral Molecular Biology, Biochemistry and Molecular Biology, and Ophthalmology, Oregon Health Sciences University, Portland, Oregon; 2 Laboratory of Biology, Senju Pharmaceutical Co., Ltd., Kobe, Japan; 3 University of Delaware, Department of Biological Sciences, Newark, Delaware; 4 Genethon, Evry, France; and 5 Centre National de Genotypage, Evry, France.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
PURPOSE. To determine the influence of specific regions within Lp82 calpain on protein stability, enzymatic activity, and localization within lens and to test the influence of an Lp82 knockout mouse on normal maturational proteolysis in lens.

METHODS. DNA constructs for Lp82 and Lp82-related proteins were subcloned into the pcDNA 3.1 vector. The constructs contained a substitution of the novel sequence (NS) region from p94 for the AX1 N-terminal region of Lp82 and insertions of the p94 IS1 and IS2 regions into Lp82. Transient expression of these Lp82-related proteins was performed in COS-7 mammalian cells. Immunoblotting and casein zymography were used to measure protein stability and enzymatic activity of the expressed proteins. Homologous recombination was used to knock out p94 gene expression and p94 splice variants such as Lp82 and Lp85 in the lenses of 10-day-old mice. Confocal microscopy revealed the immunohistochemical localization Lp82 and Lp85 within lens.

RESULTS. Insertion of IS1 into Lp82 resulted in a lack of stable protein and loss of enzymatic activity. In contrast, substitution of the NS region for AX1 and insertion of IS2 into Lp82 had no effect on the stability of the Lp82-related proteins. p94 knockout mice at 10 days of age exhibited a total absence of Lp82 activity in the lens but normal activity for the separate µ- and m-calpain gene products. Calcium-induced in vitro proteolysis was retarded in these Lp82/p94 knockout lenses. Lp82 and Lp85 immunostaining was intense throughout the cytoplasm of the cortical and nuclear fibers of newborn mouse lenses with little staining in the epithelium. In contrast, immunostaining for the ubiquitous m-calpain was highest in the epithelium and bow region, with much lower levels in the nucleus. The naturally occurring IS3 insert in Lp85 also promoted the association of Lp85 with the perinuclear region of the nucleated lens fibers.

CONCLUSIONS. The lack of the IS1 region in Lp82 accounts for the stability and abundance of enzymatically active Lp82 protein in rodent lenses. Conversely, the presence of the IS1 region is responsible for the lability of p94 and Rt88 calpains in muscle and retina, respectively. The insert in Lp85 may promote membrane association. A consequence of the specific loss of Lp82 in the lens may be to retard normal maturational proteolysis.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The calpain superfamily of cysteine proteases contains the following: the ubiquitous calpains (m- and µ-calpains),1 2 tissue-specific calpains (muscle calpain-3, also termed p94,3 stomach calpains nCl-2 and nCl-2',4 digestive tubule calpain nCl-4, lens calpain Lp82,5 and retina calpain Rt886 ), and atypical calpains. The tissue-specific calpains differ from ubiquitous calpains in their modes of expression as well as the state in which they exist.7 The canonical four-domain structure for the catalytic subunit for many calpains is comprised of domain I (membrane binding, autolytic activation), domain II (cysteine catalytic site), domain III ("electrostatic switch"), and domain IV (calcium-binding sites).8 9 The functions of specific regions within some of these domains are unknown or only speculated. For example, p94 contains three unique insert regions. The function of the novel sequence (NS) on the N terminus is unknown, insert region 1 (IS1) within the catalytic domain II contains the sites for autolysis, but its role in activation is unknown, and region 2 (IS2) between domains III and IV contains a putative nuclear localization signal and is the binding site between p94 and the giant muscle protein connectin (titan).10 The detailed physiological significance of this binding to connectin is unknown, but it may stabilize p94.

Recently, a naturally occurring splice variant of p94, called Lp82, was discovered in rodents, cows, guinea pigs and rabbits.11 This p94 variant was lens specific; Lp82 was abundant and stable in nonhuman lenses and is responsible for some proteolysis and cataract formation in rodents.12 13 Lp82 lacks the IS1 and IS2 regions and contains a complete substitution of a different N terminus (termed AX1 [alternative exon 1]) for the NS region of p94. Lp82 may thus be useful for studies to elucidate the functions of specific regions in p94. Some studies have attempted to address the functions of the NS, IS1, and IS2 regions in p94 by transient expression of mutants in COS cells. For example, substitution of AX1 from Lp82 into p94 did not affect its autolytic activity, but deletion of IS1 or IS2 region leads to stable expression of p94-related proteins.14 However, a similar study of Lp82 mutants has not been performed. Such data would be important for understanding the role of the Lp82 structure in enzymatic activity in the lens. Thus, one of the purposes of the experiments reported below was to express various structural mutants of Lp82 in COS-7 cells and to determine the effects of these mutations on protein stability and enzyme activity.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Construction of Lp82-Related Expression Plasmids
Isolation of a rat Lp82 cDNA was reported previously.5 A DNA fragment corresponding to the full-length open reading frame of Lp82 was prepared by PCR using specific primers with restriction site overhangs. After digestion with XbaI and BamHI, the DNA fragment was subcloned into a mammalian expression vector pcDNA 3.1 (Invitrogen, San Diego, CA). The Lp82 expression plasmid pcDNA3.1–82 was further modified by conventional recombinant DNA techniques to create the Lp82-related constructs depicted in Figure 1 . This resulted in the substitution of the AX1 sequence of Lp82 with the NS sequence from p94 (construct 2, p3.1–82/NS) and insertion of p94 IS2 into Lp82 (construct 4, p3.1-82+IS2). cDNA cloning of a rat retina-specific calpain, termed Rt88, was reported elsewhere.6 Rt88 is also a naturally occurring splice variant of p94 with nearly identical DNA sequence to Lp82, except Rt88 retains exon 6 (coding for IS1 region) of p94. A DNA fragment containing the full-length open reading frame of Rt88 was prepared by the PCR method and subcloned into the vector pcDNA3.1 to generate IS1 insertion plasmid (construct 3, p3.1-82 + IS1).



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  		Figure 1. Schematic diagram showing the four Lp82-related cDNA constructs (1–4) inserted in the mammalian expression vector pcDNA 3.1 and expressed in COS-7 cells and the structure of native Lp85. The numbers on the left designate lanes in Figures 2 3 4 . Calculated molecular masses and the presence (+) or absence (-) of observed enzymatic activity are summarized to the right. Top: the bar drawing indicates the sequence of the chimeric peptide synthesized from the sequences around the IS2 region. The peptide was used to generate an antibody reactive against proteins from constructs 1 to 4.

 
Expression of Lp82-Related Proteins in COS-7 Cells
The constructs described above were transfected into COS-7 cells (10 µg DNA/100-mm dish) using LipofectAMINE (Life Technologies, Rockville, MD). Cells were harvested 72 hours after transfection. Total RNA was extracted in TRIzol LS reagent (0.3 ml/3.5 cm diameter dish) according to the manufacturer’s instructions (Life Technologies). Total soluble proteins were obtained by first extracting into buffer A (20 mM Tris [pH 7.4], 1 mM EGTA, and 2 mM DTE), followed by centrifugation at 13, 000g for 20 minutes at 4°C. RT-PCR, immunoblotting, and casein zymography of these samples were performed as described below.

Reverse Transcription–Polymerase Chain Reaction
RT-PCR was performed on mRNAs from transfected cells using a pair of gene-specific primers on either side of the IS1 region (upstream primer, 703GGG TGA CAG GTT TTT TGA GAT CAA GGA729 and downstream primer, 1097GAT CTC CAG CTT TGT GAA ATG GTA GAC AAA1068. The primers were common to all Lp82 constructs, and they were based on the cDNA sequence of rat Lp82.5 Five micrograms of total RNA extracted from COS-7 cells transfected with various mutant Lp82 constructs was reverse-transcribed using an oligo-dT primer (Life Technologies). One microliter of the RT reaction mixture was subsequently transferred to a PCR reaction tube containing one Ready-To-Go PCR bead and 0.2 µM of each primer. PCR products were separated and visualized on 1.5% ethidium bromide–stained agarose gels, and Polaroid pictures (type 667; Cambridge, MA) were scanned and digitized with a flatbed scanner (Apple Color One, Cupertino, CA).

Native-PAGE, SDS-PAGE, and Immunoblotting
Proteins were separated on precast 1.0-mm-thick, 8 x 8 cm, 8% polyacrylamide mini-gels (Novex, San Diego, CA) or separated on native polyacrylamide gels without SDS. Immunoblotting was performed by electrotransferring the proteins from the mini-polyacrylamide gels to PVDF membrane at 30 V (constant) for 90 minutes at 4°C. The membranes were then incubated in TBS supplemented with 5% nonfat dry milk and 0.05% Tween-20 at room temperature for 1 hour with shaking. An affinity-purified polyclonal antibody made against a synthetic peptide spanning the deleted IS2 region in rat Lp82 termed 21Q12 (Fig. 1) was used at 1:1000 dilution, and immunoreactivity was visualized with alkaline phosphatase conjugated to anti-rabbit IgG secondary antibody and BCIP/NBT (Bio-Rad, Hercules, CA). Images of immunoblots were digitized on a flatbed scanner.

Casein Zymography
Casein (0.05%) was copolymerized in a 10% acrylamide separating gel (375 mM Tris-HCl, pH 7.4). The stacking gel was 4% acrylamide in 125 mM Tris-HCl (pH 6.8). After a 15-minute prerun, 100 µg of soluble proteins was loaded and electrophoresed at 125 V (constant) for 2 hours at 4°C in running buffer (25 mM Tris base, 192 mM glycine, 1 mM EGTA, and 1 mM DTT, pH 8.3). The gel was then incubated with slow shaking overnight at room temperature in calcium incubation buffer (20 mM Tris-HCl, 10 mM DTT, 2 mM calcium, pH 7.4). Gels were then stained in 0.05% Coomassie Brilliant Blue for 45 minutes and destained (40% methanol, 10% acetic acid in distilled water) for 60 minutes. Bands of caseinolysis appeared white against a stained background.

Tissue Collection
Lenses without decapsulation were isolated from 10-day-old control and p94 knockout Sv mice (Genethon, Evry, France; Richard I and Beckmann JS, et al., unpublished results) in buffer A. All animals were handled in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Soluble proteins were obtained by centrifugation of the lens homogenate at 13,000g for 20 minutes at 4°C. Protein concentrations were determined using the Bio-Rad dye-binding reagent with BSA as standard.

In Vitro Activation of Calpains
The total soluble proteins from whole lenses from the mice as described above were incubated at 30°C overnight in buffer containing 20 mM Tris (pH 7.4), 1 mM EGTA, 1 mM EDTA, 2 mM DTE, and 1.5 mM Ca2+. Degradation of crystallins was detected by SDS-PAGE as described above.

Immunohistochemistry on Lenses
For immunohistochemistry, unfixed lenses were embedded in TFM (Triangle Biomedical, Durham, NC) and 15-µm frozen sections were prepared. The sections were collected on Colorfrost Plus glass slides (Fisher Scientific; Pittsburgh, PA) and stored at -80°C until use. The slides were fixed in 1:1 acetone methanol at -20°C for 10 minutes and then allowed to air dry. Calpain proteins were detected with the antibodies described below. The primary antibodies, Lp82, Lp85, and m-calpain-2520, were diluted 1:200 in PBS with 1% BSA.11 12 The bound, nonfluoresceinated antibodies were visualized after incubation with the appropriate secondary antibody (Alexa Fluor 568 anti-rabbit IgG conjugated; 1:50 dilution in PBS with 1% BSA; Molecular Probes, Eugene, OR), and the cell nuclei were detected by counterstaining with SYTO-13 (1:1000 dilution in PBS; Molecular Probes). The endoplasmic reticulum was detected with Con A Alexa 594 conjugated (1:500 dilution; Molecular Probes). Slides were coverslipped, and 50 µl of mounting media (9.25 mM p-phenylenediamine in 90% glycerol with PBS) was added to each slide.15 The slides were stored at -20°C until confocal microscopy was performed on a Zeiss 510 LSM confocal microscope (Thornwood, NY) configured with an argon/krypton laser (488 and 568 nm excitation lines).

Immunofluorescence with Cell Lines
Maintenance and preparation of the {alpha}TN4–1 cells were followed as described previously.16 {alpha}TN4–1 cells (1.0–1.2 x 105) were inoculated on an eight-chamber, 1.5-mm cover glass for 20 hours. Cells were treated with ionomycin (Sigma, St. Louis, MO) for 30 minutes and then fixed with 4% paraformaldehyde in PBS for 10 minutes. The chambers were rinsed three times with PBS and permeabilized for 5 minutes with 0.2% Triton-100 in PBS. The chambers were rinsed three times with PBS and blocked with 3% BSA in PBS for 75 minutes. The primary antibody was incubated (dilution 1:200) in each chamber with 3% BSA in PBS overnight at 4°C with shaking. Chambers were rinsed three times with 3% BSA in PBS for 10 minutes. The secondary antibody was added at 1:200 dilution in PBS with 3% BSA (Alexa Fluor 568 anti-rabbit IgG conjugated; Molecular Probes), and the cell nuclei were detected by counterstaining with SYTO-13 (1:1000 dilution in PBS; Molecular Probes) for 60 minutes. The chambers were rinsed three times with 3% BSA in PBS for 10 minutes. The PBS was removed, and two drops of SlowFade Component B (S-2828; Molecular Probes) was added just before scanning on a Zeiss 510 LSM confocal microscope configured with an argon/krypton laser (488 and 568 nm excitation lines).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Expression of Lp82-Related Proteins and Their Stability
To determine whether Lp82-related mutant constructs (Fig. 1) were transfected and expressed in COS-7 cells, RT-PCR using gene-specific primers on either side of the IS1 region was performed. PCR products at the expected sizes for each of the four Lp82-related constructs were observed (Fig. 2) , suggesting appropriate mRNA expression in these cells.



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  		Figure 2. Ethidium-bromide–stained agarose gels showing results of RT-PCR using primers on either side of the IS1 region for Lp82-related constructs expressed in COS-7 cells. Arrows, migration positions (expected size for constructs 1, 2, and 4 = 396 bp; construct 3 = 540 bp). C, COS cells without transfection; V, COS-7 cells transfected with the pcDNA3.1 vector only; M, a lane containing 100-bp molecular size standards.

 
Subsequent immunoblotting of the proteins from COS-7 cell lysates expressing intact Lp82 revealed a band migrating at 82 kDa, representing the Lp82 protein (Fig. 3 , construct 1, arrowhead). The antibody used for all immunoblotting was made against a synthetic peptide comprised of the two ends surrounding the deleted IS2 region in Lp82 (Fig. 1 , top). Thus, the antibody was also capable of detecting Lp82-related isoforms with or without an intact IS2 region. Under the conditions used, a protein band representing Lp82 with the NS region from p94 substituted for the AX1 region was observed at the expected migration position of 84 kDa (Fig. 3 , construct 2, lined arrow). Also, a prominent band for Lp82 with an inserted IS2 region was observed (construct 4, lined arrow). However, when the IS1 region was inserted into Lp82, no appropriate protein band was detected (construct 3), and no degradation products were observed above 35 kDa. Theseresults suggested that the IS1 region and not the NS or IS2 regions caused instability of the Lp82-related proteins.



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  		Figure 3. Immunoblot for Lp82-related proteins expressed in COS-7 cells. Construct numbers refer to the constructs in Figure 1 ; arrowhead, wild-type Lp82; lined arrows, Lp82 mutants indicate migration positions of proteins overexpressed compared with proteins from the vector alone (lane V). Molecular weight markers in lane M are expressed as kDa.

 
Enzymatic Activity of Lp82-Related Proteins
Along with the endogenous µ- and m-calpains in COS-7 cells, zymography detected a prominent band of Lp82 caseinolytic activity in cells transfected with construct 1 (Fig. 4 , lane Lp82) but not in the cells transfected with vector alone (Fig. 4 , lane V). Substitution of the NS region for the AX1 region in Lp82 (construct 2) did not reduce caseinolytic activity (Fig. 4 , lane + NS). Caseinolytic activity was detected for construct 4, containing the IS2 insert (Fig. 4 , lane + IS2), but this activity was less than for wild-type Lp82. No caseinolytic activity was detected from the cells transfected with construct 3 containing IS1 region, except for the endogenous µ- and m-calpain activities in these cells (Fig. 4 , lane + IS1). Combined with the protein stability data above, these data suggested that the presence of the IS1 region in Lp82 structure led to the rapid breakdown of the protease in COS cells before casein zymograms were run.



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  		Figure 4. Casein zymography of Lp82-related proteins expressed in COS-7 cells (first five lanes from the left). Large arrowhead, a white band of caseinolysis for wild-type Lp82 (construct 1); smaller arrows, white bands of caseinolysis for constructs 2 and 4. On native PAGE gels, the Lp82-related constructs do not run according to molecular weights, but they migrated near native Lp82 from rat lens (lane Lens). The migration positions for other calpains are indicated on the right and identify the endogenous µ- and m-calpains ubiquitous in COS-7 cells and rat lens.

 
Less Lp82-Induced Proteolysis in p94 Knock Out Mice
At 10 days of age, Lp82 is the dominant form of calpain activity in the normal control mouse lens (Fig. 5B , lane C). The data above indicated that the splice deletion of the IS1 region (and IS2 to a lesser extent) during processing of p94 mRNA contributed to the stability and abundance of Lp82 in rodent lenses. Lp82-induced proteolysis is postulated to function during lens development and during the normal maturation of rodent lenses after birth. To test these ideas, lenses from knockout mice harboring a defective gene for p94 were analyzed. Complete loss of Lp82 protein expression as well as Lp82 activity was observed in the lenses from p94 knockout mice (Figs. 5A 5B , lane KO). This was further proof that Lp82 is a splice variant of the parent p94 gene. µ- and m-calpains are derived from different genes, and their activities were the same in knockout and control lenses (Fig. 5B) . Thus, Lp82 deficiency did not cause obvious compensatory overexpression of µ- and m-calpains in the lens.



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  		Figure 5. Immunoblot (A), casein zymogram (B), and SDS-PAGE analysis (C) of the total soluble proteins from 10-day-old control mice (C) and knockout mice with deletion of the p94 gene (KO). Lanes designated -Ca contained no free calcium, whereas lanes marked +Ca were incubated overnight in 1.5 mM Ca2+. Arrows, protein bands showingchanges.

 
SDS-PAGE was also performed to compare normal maturational proteolysis of crystallins in the control and knockout lenses. Crystallins may have been slightly more fragmented in the control lenses than in knockout mice at 10 days of age (Fig. 5C , lanes 1 and 3). Ca2+ was then added to the two lens samples to artificially accelerate this maturational proteolysis (Fig. 5C , lanes 2 and 4). Incubation with calcium enhanced proteolysis in the control samples but not in the knockout lenses.

Influence of Domain IV Insert on Localization of Lp82 in Lens
Unlike some of the "synthetic" constructs used to transfect COS cells described above, Lp85 is a naturally occurring rodent lens isoform of Lp82. Lp85 contains a unique 28 amino acid insert termed IS3 (retained intron 18) in the calcium-binding domain IV (Fig. 1) . Our previous studies showed that this IS3 insert did not interfere with caseinolytic activity and that the Lp85 protein was stable.11 The present study attempted to further explore the influence of IS3 on protein localization in lens by using immunohistochemistry. Two antibodies were used. The Lp85 antibody was made against the unique 28 amino acid insert peptide of IS3. The other antibody was the 21 Q antibody used above, which reacts with Lp82 isoforms. Confocal microscopy with the Lp82 antibody revealed that Lp82 was located in the cytoplasm of cortical and nuclear fibers of the newborn mouse lens (Fig. 6A , red stain). The epithelium was essentially negative for Lp82. No Lp82 was found associated with the cell nuclei (Fig. 6D) . Although this antibody could also detect Lp85 when it was used in the immunoblotting of lens total soluble proteins, the results described below suggest that staining in Figures 6A and 6D was for Lp82.



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  		Figure 6. Confocal micrographs of newborn mouse lens showing immunohistochemical localization of Lp82 (A, D), Lp85 (B, E), and negative control lacking primary antibody (C, F). Red, positive staining for Lp82 or Lp85 proteins; green, DNA staining in the cell nuclei. b, lens bow region; c, cornea; e, lens epithelium; i, iris; pf, primary fibers; sf, secondary fibers. Approximate magnifications, (top) x70; (bottom) x441.

 
Staining with an Lp85-specific antibody showed that the 28 amino acid insert in Lp85 altered the distribution of Lp85 in lens compared with Lp82. That is, although Lp85 was found in the cytoplasm of denucleated fibers (Fig. 6B) , Lp85 was concentrated in the peri-nuclear region of the nucleated cortical fibers. (Fig. 6E) . Triple staining of this region for Lp85, DNA, and an endoplasmic reticulum marker (Con A) also showed that Lp85 was concentrated around the nucleus but was not associated with the endoplasmic reticulum (data not shown). These observations indicated that the IS3 insert probably promotes association of Lp85 with membranes of the nuclear envelope.

In contrast, immunohistochemical localization of an ubiquitous calpain, m-calpain, was performed and revealed a distribution pattern different from that of Lp82. m-Calpain was concentrated in the cytoplasm of both the lens epithelium and fiber cells in the outer regions of the lens (Figs. 7A 7B ). However, in the lens fibers, significant m-calpain was found associated with the nuclear envelope (Fig. 7B , arrowhead). Cytoplasmic localization of m-calpain was even more clearly visualized in cultured {alpha}TN4–1 mouse epithelial cells (Fig. 7C) . Treatment of {alpha}TN4–1 cells with 0.1 to 100 µM ionomycin leads to a redistribution of immunoreactivity, presumably associated with autolytic activation caused by elevated intracellular calcium (Fig. 7D) .



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  		Figure 7. Confocal micrographs showing immunohistochemical localization of m-calpain in newborn mouse lens (A, B) and in {alpha}TN4–1 mouse lens epithelial cells with no calcium ionophore (C), with 10 µM calcium ionomycin for 30 minutes (D). Red, positive staining for m-calpain protein; green, DNA staining in the cell nuclei; b, lens bow region; c, cornea; e, lens epithelium; i, iris, pf, primary fibers; sf, secondary fibers; t, transition zone; arrowhead, punctate distribution of m-calpain on the fiber cell nuclei. Approximate magnifications, (A) x90; (B, C, D) x630.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The major finding of the present investigation was that certain regions in lens-specific calpain Lp82 control protein stability and distribution within lens. In particular, insertion of IS1 caused breakdown of Lp82 protein and loss of enzymatic activity (Figs. 3 and 4 , + IS1). The IS1 region, which is partially encoded by exon 6, is located in the proteolytic domain II. We speculate that the protein was degraded soon after translation because all other transcripts were translated in our COS cell system (Fig. 3) , the Lp82 + IS1 construct was also labile in the baculovirus expression system,6 and p94 expressed in COS cells was very labile.17 In p94, the IS1 region was shown to be the location of three autolysis sites.17 Previous failures to detect and purify p94 protein from skeletal muscle led to the conclusion that the IS1 region was responsible for protein instability. The present data indicate that this is also true when IS1 is inserted in a lens-specific calpain, Lp82.

The splice deletion of the IS1 region in Lp82 (Fig. 1 , construct 1) helps explain the abundance and stability of this protease in lens. Lp82 is readily detected in a variety of lenses including those from rats, mice, cows, rabbits, and pigs11 but not in humans.18 In mice, Lp82 is the dominant form of calpain.19 Conversely, rapid autolysis near the IS1 region probably explains why Rt88, containing the IS1 region (Fig. 1 , construct 3), was not detectable in our system. The observation that very little Rt88 protein was present in retina samples (whereas mRNA is abundant) is likely due to the presence of the IS1 region in the molecule, causing rapid degradation of the protein in vivo.6

Our hypothesis is that the presence or absence of the IS1 region is related to a specific function in the tissue-preferred location. In lens, a stable Lp82 protease may be needed for long-term proteolysis of crystallins during lens development and maturation. This was supported by the fact that less calcium-induced proteolysis of crystallins in Lp82-deficient knockout mice was observed (Fig. 5C) . This was expected because, in addition to producing a unique cleavage site on the C terminus of {alpha}A-crystallin,20 Lp82 activity in maturing rat lens is roughly equal to m-calpain activity. On the other hand, a quickly degraded Rt88 may be beneficial for a short-term signaling event in the retina.

Substitution of the NS region for AX1, and insertion of IS2 into Lp82, had no effect on the stable expression of the Lp82-related proteins in COS cells (Figs. 3 , + NS and + IS2). Insertion of IS2 reduced enzymatic activity somewhat (Fig. 4 , + IS2). The functions of the NS and IS2 regions are unknown. In previous reports, IS2 was presumed to have an important role in the rapid autolysis of p94.21 However, the present data indicated that the presence of the IS1 region exerted a much greater influence on stability of Lp82.

The present studies also suggested that another insert, the 28 amino acid insert located in the domain IV of the calmodulin-like, calcium-binding region of Lp85, affected localization of the protease. Confocal microscopy using an Lp85 antibody revealed that Lp85 was localized around the nuclear membrane in the cortical region of lens in addition to its cytoplasmic localization. In contrast, splice deletion of the insert to form Lp82 caused a totally cytoplasmic distribution of the protein in lens. The exact biological function of the observed association of Lp85 with the epithelial and young fiber cell nuclear membrane is currently unknown. We speculate that the Lp85 insert may perform a binding function similar to the situation for the IS2 region in muscle p94. The IS2 region was shown to be a site for binding of p94 to a gigantic myofibrillar protein called connectin.10 The detailed physiological significance of this binding to connectin is unknown, however, but mutations in p94 gene are responsible for human limb girdle muscular dystrophy (type 2A).22 Discovery of a similar binding partner for Lp85 will be important because this may shed new light on the in vivo function and control of this protease.

Our previous studies showed that the mRNA levels as well as enzymatic activities for m-calpain were highest in the outer regions of young rat lens.23 24 The present study using immunohistochemistry showing distribution of m-calpain in lens corresponds well to these previous observations (Fig. 7A) . The outer regions of lens including epithelium and young fiber cells are the sites of rapid fiber cell differentiation during lens maturation. The high level of m-calpain in the outer regions may be required for cell remodeling during differentiation.

In a wide variety of young rat models of cataract, the common underline mechanism is believed to be truncation of crystallins by calpain after increased lens calcium. This is followed by insolubilization of the truncated crystallins, light scatter, and opacity. We recently reported that Lp82, along with m-calpain, played a major role in the formation of mouse cataract induced by either calcium ionophore A23187 in cultured lens or BSO in vivo.20 The p94/Lp82 knockout mouse provided a useful model to study the consequences of the specific loss of Lp82 in lens. Although only slightly less proteolysis of crystallins was observed in vivo in the 10-day-old p94/Lp82-deficient mice, in vitro proteolysis was obviously retarded when calcium was added to these samples (Fig. 5C) . The long-term consequences of the Lp82 deficiency on normal maturational proteolysis in the lens is thus under active investigation.


   Acknowledgements
 
The authors thank Muriel Herasse, Carinne Roudaut, the Association Francaise contre les Myopathies (AFM), and Kirk Czymmek of the University of Delaware core microscopy facility for technical assistance.


   Footnotes
 
Supported in part by National Institutes of Health Grants EY05786 (TRS) and EY112221 (MKD).

Submitted for publication July 14, 2000; accepted August 17, 2000.

Commercial relationships policy: E (CF, MA); F,R (TRS); N (all others).

Corresponding author: Thomas R. Shearer, Oregon Health Sciences University, 611 SW Campus Drive, Portland, OR 97201. shearert{at}ohsu.edu


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Emori, Y, Kawasaki, H, Sugihara, H, Imajoh, S, Kawashima, S, Suzuki, K. (1986) Isolation and sequence analyses of cDNA clones for the large subunits of two isozymes of rabbit calcium-dependent protease J Biol Chem 261,9465-9471[Abstract/Free Full Text]
  2. Croall, DE, Demartino, GN (1991) Calcium-activated neutral protease (calpain) system: structure, function, and regulation Physiol Rev 71,813-847[Free Full Text]
  3. Sorimachi, H, Imajoh-Ohmi, S, Emori, Y, et al (1989) Molecular cloning of a novel mammalian calcium-dependent protease distinct from both m-and mu-types. Specific expression of the mRNA in skeletal muscle J Biol Chem 264,20106-20111[Abstract/Free Full Text]
  4. Sorimachi, H, Ishiura, S, Suzuki, K. (1993) A novel tissue-specific calpain species expressed predominantly in the stomach comprises two alternative splicing products with and without Ca(2+ )binding domain J Biol Chem 268,19476-19482[Abstract/Free Full Text]
  5. Ma, H, Fukiage, C, Azuma, M, Shearer, TR (1998) Cloning and expression of mRNA for calpain Lp82 from rat lens: splice variant of p94 Invest Ophthalmol Vis Sci 39,454-461[Abstract/Free Full Text]
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