peptidases play an important role in cataractogenesis: an immunohistochemical study on lenses...

The Histochemical Journal 33: 511–521, 2001.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

Peptidases play an important role in cataractogenesis: An immunohistochemicalstudy on lenses derived from Shumiya cataract rats

Hui Zhang1,3, Yishio Yamamoto2, Seigo Shumiya4, Mitoshi Kunimatsu5, Katsuji Nishi2, Iwao Ohkubo3,∗ &Kazutaka Kani1

1Department of Ophthalmology, 2Department of Legal Medicine, 3Department of Medical Biochemistry,Shiga University of Medical Science, Seta Otsu 520-2192, Japan4Department of Laboratory Animal Science, Division of Gerotechnology Research, Tokyo Metropolitan Institute ofGerontology, Itabashi-ku, Tokyo 173-0015, Japan5Second Department of Biochemistry, Nagoya City University Medical School, Mizuho-ku, Nagoya 467-8601, Japan

∗Author for correspondence

Received 19 September 2001 and in revised form 26 November 2001

Summary

The role of proteolytic enzymes in Shumiya cataract rats in alterations to lens proteins during cataract formation was studiedimmunohistochemically using antibodies against exopeptidases, such as lysosomal dipeptidyl peptidase II (DPP II), cytosolicdipeptidyl peptidase III, and soluble and membrane-bound alanyl aminopeptidases, and against cytosolic endopeptidases suchas µ- and m-calpains, and 20S proteasome. αB-crystallin was detected as a proteolytic marker in the lenses. A constantimmunoreactivity against all the antibodies employed was observed in the lens epithelium independent of the strain and ageof the rats. A weak immunoreactivity against exo- and endopeptidases and an intense reactivity against αB-crystallin wereobserved in the lens fibres of control rats at all ages. The immunoreactivity of these peptidases in lens fibres increased with agein cataract rats, but that of αB-crystallin decreased. No reactivity against exo- and endopeptidases was seen in the perinuclearregion of lenses of control rats at all ages or in Shumiya cataract rats at 8 and 10 weeks of age, but an intense reactivity againstthese peptidases was observed in the lens perinuclear region of lenses in cataract rats at 12 and 14 weeks of age. αB-crystallinimmunoreactivity was observed with ordered striations in the lens perinuclear region of all control rats whereas the striationsin this area of cataract rat lens were disorganized. Membrane-bound alanyl aminopeptidase was detected feebly in the lensepithelium and fibres of both types of rat at all weeks of age. These findings indicate that exo- and endopeptidases, exceptfor membrane-bound alanyl aminopeptidase, are expressed intensively and are age-dependent. Conversely, the amount ofαB-crystallin decreased with age in lens fibres of cataract rats. Calpains (µ- and m-), 20S proteasome, dipeptidyl peptidases IIand III and soluble alanyl aminopeptidase are thought to induce lens opacification kinetically during cataract formation inShumiya cataract rats through the intracellular turnover of lens proteins.

Introduction

Cataracts cause severe visual impairment in a large num-ber of people, especially after middle age (Mizuno et al.1992). It is widely accepted that lens proteins are damagedby infrared and ultraviolet rays, various oxidative speciesand inherited factors. The diminished ability to degrade thedamaged proteins may cause precipitation and subsequentcataract formation (Taylor & Davies 1987). The degradationof α-, β-, and/or γ -crystallins, which are the main water-soluble proteins in the eye lens, has been demonstrated insevere cataracts (Barber 1973) and selenite nuclear cataract(Shearer et al. 1997), in congenital cataracts of Nakano mice(Piatigorsky et al. 1978) and Shumiya cataract rats (SCRs)(Inomata et al. 1997). It has been proposed that variousendogenous peptidases in the lens such as calpains (Inomataet al. 1997) and proteasome (Andersson et al. 1998) may

be involved in the proteolytic modification of lens proteinsduring cataract formation.

In our previous studies, we purified some exopeptidasessuch as dipeptidyl peptidase II (DPP II), dipeptidyl pepti-dase III (DPP III), alanyl aminopeptidase N (AAP-N) andpuromycin-sensitive alanyl aminopeptidase (AAP-S) (Huanget al. 1996, Ohkubo et al. 1999, Huang et al. 1997, Yamamotoet al. 1998) and endopeptidases including 20S proteasome(Ohkubo et al. 1991) and µ- and m-calpains (Ishiguro et al.1987, Onizuka et al. 1995, Saito et al. 1999). The physico-chemical properties of the peptidases were characterized,and we have prepared polyclonal and monoclonal antibod-ies against these enzymes. However, the localization of thepeptidases in the lens during normal ageing and cataractousprocess has not yet been elucidated.

Here, we examined the distribution and reactivity offour exopeptidases and two endopeptidases in normal and

512 H. Zhang et al.

cataract lenses immunohistochemically in order to investi-gate whether the peptidases are involved in the induction oflens opacification during cataract formation.

Materials and methods

Animals

Breeding stock SCR, which have a hereditary cataract andin which lens opacity appears spontaneously in the nuclearand perinuclear portions at 11–12 weeks of age, were used(Shumiya 1995), Wistar rats were used as normal controls.SCRs and Wistar rats were kept in an air-conditioned, cleanroom and handled in compliance with the guiding principlesin care and use of animals in our university. The ages ofthe rats examined were 8-, 10-, 12- and 14-weeks old and10 each of SCRs and Wistar rats were used at each age forimmunohistochemical experiments. The rats were perfusedthrough the left cardiac ventricle with 10 mM phosphate-buffered saline (PBS) followed by 10% formalin under deepanaesthesia induced by intraperitoneal administration of pen-tobarbital (40 mg/kg). The eyes were removed and postfixedwith 10% formalin for 20 h, immersed in water for 24 h, dehy-drated in ethanol, substituted with benzene, and embedded inparaffin wax.

Antibodies

Antibodies were prepared against the following enzymes:purified lysosomal DPP II from porcine seminal plasma(Huang et al. 1996), DPP III from rat liver cytosol (Ohkuboet al. 1999), AAP-S from rat liver cytosol (Yamamotoet al. 1998), membrane-bound AAP-N from human semi-nal plasma (Huang et al. 1997), and 20S proteasome fromhuman red cells (Ohkubo et al. 1991). Two specific antibod-ies with no cross-reactivity against human µ- and m-calpainswere also prepared. We raised an antibody against apeptide (N -acetyl-SEETPVYCTGVSAQVQKQRARELG)of µ-calpain (Onizuka et al. 1995) and a mono-clonal antibody against a peptide (MAGIAAKLAK-DREAAEGLGSHERAIKYLNQD) originating from humanm-calpain (Saito et al. 1999), respectively. A polyclonal anti-body against the native form of αB-crystallin was purchasedfrom Cosmo Bio (Tokyo, Japan).

Immunohistochemistry

Paraffin sections (4-µm thick) were cut using a microtome,and mounted on slides. After deparaffinization and rehydra-tion, the sections were immersed in methanol containing 3%hydrogen peroxidase for 10 min to quench endogenous per-oxidase activity and then treated in PBS containing 10% nor-mal sheep serum for the polyclonal antibody and in PBScontaining 10% rabbit serum for the monoclonal antibody.The sections were incubated with a solution of the primaryantibody diluted in PBS (dilutions were 1 : 1,000–1 : 10,000

for polyclonal antibodies and 1 : 10,000 for monoclonal anti-body) overnight at 4 ◦C. Control sections were incubated withPBS containing 10% sheep serum without primary antibodyand with PBS containing the mixture of the antibody andthe corresponding enzyme. The sections were incubated in asolution of biotin-conjugated IgG for 30 min and were incu-bated with streptavidin–biotin preformed complex for 30 min.The reaction was visualized by incubation in PBS containing0.03% 3,3′-diaminobenzidine and 0.3% hydrogen peroxidase(Histofine SAB-Po kit, Nichirei, Tokyo, Japan). The slideswere washed three times with PBS for 10 min at each inter-val of process and counterstained with haematoxylin. Thereactivity of the peptidases in the lenses was determined bylight microscopical observation based on the reactivity in 20randomly selected sections.

Results

Localization of exopeptidases

Among the four exopeptidases tested, cytosolic DPP III(Figure 1A–H) and AAP-S (Figure 2C and D) and lysosomalDPP II (Figure 2A and B) were detected with weak reactivityin the lens epithelium from both types of rat at all ages and thedensity of their reactivity was constant. DPP III, DPP II andAAP-S were also observed with weak and constant reactivityin lens fibres of control rats (Figure 1A, C, E and G; Figure 2Aand C), but the reactivity in the lens fibres from SCRs wasstronger than that of control rats at all ages (Figure 1B, D, Fand H; Figure 2B and D). The reactivity of DPP III in lensfibres of SCRs increased from moderate to intensive at 12and 14 weeks of age, and the reactive area was extended toinclude the perinuclear region where the opacity of lens hadfirst developed (Figure 1F and H). Although no reactivity ofDPP III, DPP II and AAP-S was observed in the lens perinu-clear region from control rats at all weeks and from SCRs at8 and 10 weeks of age, moderate or intensive reactivity of theenzymes appeared in this region in SCRs at 12 and 14 weeksof age (Figure 1F and H; Figure 2B and D). Membrane-boundAAP-N was detected weakly in lens epithelium, feebly in lensfibres, and was not detectable in the lens perinuclear regionin both control rats and SCRs at all weeks of age (Figure 2Eand F). The four enzymes could not be detected in the lensnuclear region of both types of rat at all weeks of age (datanot shown).

Localization of endopeptidases

Before detecting µ- and m-calpains in lenses, we prepared amono-specific antibody against a peptide originating fromhuman µ-calpain and also raised a monoclonal antibodyagainst a peptide originating from human m-calpain asdescribed in the Materials and methods section.

µ-Calpain was detected with moderate reactivity in thelens epithelium from both types of rats at all weeks of age(Figure 4A and B), and 20S proteasome was also detected

Role of peptidases in cataractogenesis 513

Figure 1. Immuohistochemical localization of DPP III in a control rat and SCR lenses of various ages. In control lenses at 8 (A), 10 (C), 12 (E) and14 (G) weeks, weak immunoreactive staining is observed in fibres (arrow). In SCR lenses at 8 (B), 10 (D), 12 (F) and 14 (H) weeks, the immunoreactivestaining in fibres (arrow) is stronger than that of control rat lenses. At 12 and 14 weeks of age, immunoreactive staining is seen to extend markedlyinto the perinuclear region (arrowhead) where opacity has developed. Bar = 50 µm.

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Figure 2. Immuohistochemical localizations of DPP II, AAP-S and AAP-N in the control rat and SCR lenses at 12 weeks of age. The immunoreactivityof DPP II in the fibres (arrow) of the SCR lens (B) is stronger than that of control lens (A). Moreover, the immunoreactive staining is seen to extendmarkedly into the perinuclear region (arrowhead) of the SCR lens. Immunohistochemical localization of AAP-S in the control (C) and SCR (D) lensesat 12 weeks of age, the positive staining is the same as DPP II. As to AAP-N, no immunoreactive change was observed in the control (E) and SCR (F)lenses at 12 weeks of age. Bar = 50 µm.

with weak reactivity in the epithelium of both control and testrats at all ages (Figure 4C and D). On the other hand, a weakbut constant m-calpain reactivity was detected in the lensepithelium of control rats at all ages (Figure 3A, C, E and G).

In contrast, a weak m-calpain reactivity was observed in thelens epithelium of SCRs at 8 weeks of age, and the reactivitygradually decreased in the epithelium of SCRs after 10 weeksof age (Figure 3B, D, F and H).


Figure 3. Immunohistochemical localization of m-calpain in the control rat and SCR lenses of various ages. In control lenses at 8 (A), 10 (C),12 (E) and 14 (G) weeks, weak immunoreactivity is observed in fibres (arrow). In SCR lenses at 8 (B), 10 (D), 12 (F) and 14 (H) weeks of ages, theimmunoreactive staining in fibres (arrow) is stronger than that of the control rat lenses. At 12 and 14 weeks of age, immunoreactive staining is seento extend markedly into the perinuclear region (arrowhead) where opacity has developed. Bar = 50 µm.

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Figure 4. Immuohistochemical localizations of µ-calpain, 20S proteasome and αB-crystallin in the control rat and SCR lenses at 12 weeks of age.The immunoreactivity of µ-calpain in the fibres (arrow) of SCR lens (B) is stronger than that of control lens (A). Moreover, the immunoreactivestaining is seen to extend markedly into the perinuclear region (arrowhead) of SCR lens. Immunohistochemical localization of 20S proteasome inthe control (C) and SCR (D) lenses at 12 weeks of age, the positive staining is the same as µ-calpain. The immunoreactivity of αB-crystallin wasdecreased in the fibres (arrow) of SCR lenses (F) as compared with control lens (E). Bar = 50 µm.

In the lens fibres of the control rats, a weak but constantµ-calpain reactivity was detected at all ages, but the area oflens fibres with moderate reactivity gradually decreased at12 and 14 weeks of age. In contrast, a moderate or strong

m-calpain reactivity was detected in the lens fibres in SCRsat all ages (Figure 4A and B). Furthermore, a weak and con-stant µ-calpain reactivity was detected in the lens fibres ofcontrol rats at 8 and 10 weeks of age, but the area of lens


fibres with weak reactivity decreased at 12 and 14 weeksof age (Figure 3A, C, E and G). Inversely, the reactivity ofm-calpain showed an age-dependent gradual increase in thelens fibres of SCRs and the strongly stained areas of the lensfibres remained constant (Figure 3B, D, F and H).

A weak and constant 20S proteasome reactivity wasdetected in the lens fibres of control rats at all ages(Figure 4C). Moderate staining for the enzyme was presentin the lens fibres of SCRs at all ages (Figure 4D). The reactiv-ities of µ- and m-calpains and 20S proteasome in lens fibresof SCRs was stronger than those in control rats (Table 2).

No reactivity for µ- and m-calpains or 20S proteasome wasobserved in the lens perinuclear region of the control rats atall ages and of SCRs at 8 and 10 weeks of age (Figure 3A–Eand G; Figure 4A and C), while an intensive reactivity forthree enzymes in SCRs was detected at 12 and 14 weeks ofage. Opacity was also observed in this area (Figure 3F and H;Figure 4B and D).

The three enzymes were not observed in the lens nuclearregion of either type of rat at any age (data not shown).

Localization of αB-crystallin

An intenseαB-crystallin reactivity was detected in epitheliumand fibres in the lens of control rats at all ages (Figure 4E). Thereactivity of αB-crystallin in lens fibres of SCRs remainedintense until 10-weeks old. However, the reactivity in lensfibres was reduced at 12 and 14 weeks of age (Figure 4F).A weak αB-crystallin staining with ordered striations waspresent in the lens perinuclear region of all control rats atall ages (Figure 4E) whereas the striations in this area ofthe SCR lenses were disorganized at 12–14 weeks of age,and the entire lens perinuclear region of SCRs was obtained(Figure 4F).

The staining results obtained in this study are summa-rized in Tables 1–3, and are also presented in Figures 1–4.

Table 1. Immunohistochemical localization and reactivity of various exopeptidases DPP II, DPP III, AAP-S andAAP-N in control and SCR lenses.

Enzyme Age (weeks) Epithelium Fibres Perinuclear

Control Cataract Control Cataract Control Cataract

DPP III 8 + + + ++ − −10 + + + ++ − −12 + + + ++ ∼ +++ − ++ ∼ +++14 + + + ++ ∼ +++ − + + +

DPP II 8 + + + ++ − −10 + + + ++ − −12 + + + ++ − ++14 + + + ++ − ++

AAP-S 8 + + + + ∼ ++ − −10 + + + + ∼ ++ − −12 + + + ++ − + ∼ ++14 + + + ++ − ++

AAP-N 8 + + ± ± − −10 + + ± ± − −12 + + ± ± − −14 + + ± ± − −

−: no staining in any cells, ±: weak staining in some cells, +: weak staining in most cells, ++: positive staining inmost cells, +++: strong staining in most cells.

An overview of the localization and reactivity of exo- andendopeptidases in the lenses of both types of rats is shown inFigure 5.

Discussion

The transparency of the lens is dependent on both the regulararrangement of the lens fibres and the uniform distributionof lens proteins (Tardieu & Delaye 1988). The modificationof lens proteins induced by oxidation, glycation, deamida-tion and/or ageing, and the degradation of the cytoskeltoncaused by the activation of peptidases in the lens results incataract formation. Several biochemical factors such as thedegrading or cleaving of α-crystallins (A and B) and βA1-,βA4- and βB1-crystallins by m-calpain (David et al. 1993,Inomata et al. 1997, Tomohiro et al. 1997) have been reportedin the context of cataract formation. However, the mecha-nism of cataract formation still remains unclear, and there islittle information concerning detailed and age-related distri-butions of exo- and endopeptidases in the lenses of normaland cataract rats.

In our experiments, exo- and endopeptidases includingDPP III, DPP II, AAP-S, µ-calpain and 20S proteasomeshowed similar levels of detection in the lens epithelium ofboth types of rat at any age (Tables 1 and 2, Figures 1–4),while αB-crystallin, which is one of the main water-solubleproteins, is intensely immunoreactive in the lens epitheliumof both types of rat at any age (Table 3 and Figure 4E). It hasbeen suggested that the cleaving of αB-crystallin by theseenzymes may not occur in the lens epithelium, and that theseenzymes do not play a major role in cataract formation inthe lens epithelium. A membrane-bound AAP-N is thoughtto be excluded as a contributor to cataract formation sincethe reactivity of the enzyme was not observed in other areasexcept for the lens epithelium and fibres of both types of ratat any age.

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Table 2. Immunohistochemical localization and reactivity of various endopeptidases µ-calpain, m-calpain and20S proteasome in control and SCR lenses.

Enzyme Age (weeks) Epithelium Fibres Perinuclear


µ-Calpain 8 + + ++ ++ − −10 + + ++ ++ − −12 + + + ++ ∼ +++ − +++14 + + + ++ ∼ +++ − +++

m-Calpain 8 + + + + ∼ ++ − −10 + ± ∼ + + ++ ∼ +++ − −12 + ± ∼ + ± ∼ + +++ − + + +14 + ± ∼ + ± ∼ + +++ − + + +

20S proteasome 8 + + + + ∼ ++ − −10 + + + + ∼ ++ − −12 + + + ++ ∼ +++ − ++ ∼ +++14 + + + ++ ∼ +++ − ++ ∼ +++

−: no staining in any cells, ±: weak staining in some cells, +: weak staining in most cells, ++: positive stainingin most cells, +++: strong staining in most cells.

Table 3. Immunohistochemical localization and reactivity of αB-crystallin in control and SCR lenses.

Age (weeks) Epithelium Fibres Perinuclear


8 ++ ++ +++ ++ ∼ +++ + ± ∼ +10 ++ ++ +++ ++ ∼ +++ + ± ∼ +12 ++ ++ +++ ± ∼ + + ± ∼ +14 ++ ++ +++ ± ∼ + + ± ∼ +−: no staining in any cells, ±: weak staining in some cells, +: weak staining in most cells, ++:positive staining in most cells, +++: strong staining in most cells.

αB-crystallin was strongly stained in the lens epitheliumof both types of rat and lens fibres of control rats, and weaklystained with ordered striations in the lens perinuclear regionof the control rat lenses, but the striation lines in this areaof SCRs lens were disorganized at 12 and 14 weeks of age.This result indicates that the protein plays an important roleas a structural protein in the lens epithelium, fibres and peri-nuclear region. The amount of αB-crystallin was obviouslyreduced in the lens fibres in SCRs at 12 and 14 weeks of age(Figure 4F).

Endopeptidases, such as µ- and m-calpains and 20S pro-teasome, and the exopeptidase DPP III were strongly stainedin the lens fibres and perinuclear region of SCRs at 12 and14 weeks of age (Figure 4B; Figure 3F and H; Figure 4D;Figure 1F and H). Furthermore, DPP II and AAP-S showed amoderate or intense reactivity in the lens perinuclear regionof SCRs at 12 and 14 weeks of age (Figure 2B and D). Thesefindings indicate that enzymes including µ- and m-calpainsand 20S proteasome participate in cleaving αB-crystallin inthe lens fibres and perinuclear region of SCRs. In the presentexperiments, we could not determine the content of calciumions in the lenses. However, Hightower et al. (1987) reportedthat the free calcium concentration was less than 3 µM withinlens nuclear fibres of the normal control lenses, but the con-centration of calcium increased up to more than 100 µM inselenite cataract lens. Inomata et al. (1997) also indicatedthat the total calcium concentration in cataractous (opaque)

SCR lenses increased about 5-fold in comparison with that incontrol rat lenses. Our findings suggested that both calpainswere activated by Ca2+ influx into the SCR lens cells. It is pre-dicted that the elevation of calcium concentration in the lensbrings about the activation of calpains, resulting in the degrad-ing of α- and β-cystallins by both activated calpains. In fact,Inomata et al. (1997) specified the cleavage sites at C-terminalprotions of αA- and αB-crystallins and at the N-terminalportion of βB1-crystallin by activated m-calpain. Further-more, they attested the localization of cleavage products inthe SCR lenses. Shearer et al. (1997) and David et al. (1993)also indicated that the activated m-calpain in selenite nuclearcataract cleaves and releases at least 8 new fragments fromC-terminal portions ofβ-crystallins, and the insolubilized andproteolyzed α- and β-crystallins co-precipites γ -crystallins.It is suggested that the phenomenon observed in the selenitenuclear cataract similarly occurs in the lens fibres and perin-uclear region of SCR lenses.

Tomkinson (1999) proposed that the major sites for pro-teolytic degradation are lysosomes and cytosol in the cells,and also indicated that in these compartments, proteinsare sequentially degraded by endopeptidases such as cal-pains and 20S and 26S proteasomes and exopeptidasessuch as tripeptidyl peptidases, dipeptidyl peptidases, tripetideaminopeptidase(s) and aminopeptidases. Judging from ourdata, it is thought that αB-crystallin and other crystallins areessentially degraded by calpains in the SCR lenses, and then


Figure 5. Immunohistochemical localization of exo- and endopeptidases in the lenses of control rats and SCRs. A: DPP III; B: DPP II; C: AAP-S;D: AAP-N; E: µ-calpain; F: m-calpain; G: 20S proteasome; H: αB-cystallin. Upper and lower panels in each column show the lenses of control ratsand SCRs.

the degraded crystallin fragments are attacked by exopep-tidases such as DPP III, DPP II and AAP-S. With regardto the degradation of αB-crystallin, Boelens et al. (2001)reported that 20S proteasome could bind to αB-crystallinvia its C8/α7 subunit, but no strong interaction between20S proteasome and αB-crystallin was observed. They alsosuggested that the interaction between αB-crystallin andthe C8/α7 subunit might affect the assembly of proteasomecomplexes or facilitate the degradation of unfolded pro-teins bound to αB-crystallin. Murakami et al. (1990) alsoreported that lens proteasome enhanced rates of degrada-tion of hydroxyl radical modified α-crystallin. Anderssonet al. (1998) suggested that proteasome in the lens epitheliumfrom clear and cataractous human lenses cleaved the substrate

Suc–Leu–Leu–Leu–Val–Tyr–MCA, and proteolytic activityof proteasome toward the substrate was higher in lens epithe-lium from clear lenses than that from cataractous lenses,and also indicated that there was no difference in the activ-ity of the proteasome between cortical and non-corticalcataracts.

However, in our immunohistochemical experiments, 20Sproteasome antibody always showed a strong reaction towardproteasome in the fibres and perinuclear region of SCR lenses(Figure 4D). Accordingly, it is thought that the increase of20S proteasome reactivity in the fibre and perinuclear regionof SCR lenses allows both calpains to degrade αB-crystallin.Although the mechanism for increasing the expression of pro-teasomes and calpains is still unknown in the SCR lenses, the

520 H. Zhang et al.

Figure 6. Model mechanism for cataract formation associated by peptidases. The scheme was assembled with our data and the findings from paperspreviously reported (Hightower et al. 1987, Murakami et al. 1990, Inomata et al. 1997, Andersson et al. 1998, Tomkinson 1999, Inomata et al. 2000,Boelens et al. 2001).

activation of µ- and m-calpains is dependent on the increaseof calcium ion concentration into the lens cells (Hightoweret al. 1987, Inomata et al. 1997, 2000, Tomohiro et al. 1997).On the basis of our findings, it might be proposed that µ- andm-calpains, 20S proteasome, DPP III, DPP II and AAP-S par-ticipate in cataract formation in the lens fibres and perinuclearregion of SCRs at various steps. The activity of DPP III in theEmory mouse lens was elevated to a 1.5-fold higher level thanthat of cataract resistant mice (Swanson et al. 1985). The reac-tivity of DPP III in lens fibres of SCRs increased from moder-ate to intense at 12 and 14 weeks of age (Figure 1F and H). Ourfindings indicate increased levels of DPP III which are ableto cleave and release dipeptides such as Arg–Arg, Ala–Arg,Asp–Arg and Tyr–Gly from N-termini of αB-crystallin frag-ments if these dipeptide sequences are available. In contrast,the reactivity of cytosolic AAP-S observed in the lens fibresof SCR was stronger than that of control rats at all ages(Figure 2C and D). It is thought that the increased AAP-S is also able to cleave and release amino acids such asLys, Met, Arg, Ala, Leu, Phe and Tyr from N-termini ofαB-crystallin fragments if these amino acid sequences areavailable (Yamamoto et al. 1998). Although the levels ofDPP II activity in cataract lens have not been reported, theincreased antigen levels of DPP II observed in our study indi-cate that DPP II is able to release dipeptides such as Lys–Ala,Lys–Pro, Arg–Pro, Phe–Pro, Arg–Pro, Ala–Pro, Arg–Ala andAla–Ala from N-termini of αB-crystallin fragments or fromthose of β- and γ -crystallins when lysosome is disrupted in

the process of cataract formation. However, whether lysoso-mal exo- and endopeptidases participate in the cataract for-mation in SCR lenses needs further analysis.

Based on our findings and the several papers cited, wepropose a new concept of the mechanism of cataract forma-tion (Figure 6). However, further investigations are needed tocomprehend the possible role of exo- and endopeptidases inthe mechanism of cataract formation.

Acknowledgements

This work was supported in part by grants from The Ministryof Education, Science and Culture of Japan (Research Grant11670413 to K.N., Research Grant 13202056 to M.K.). Wewish to thank Mr. Takefumi Yamamoto (Central ResearchLaboratory, Shiga University of Medical Science) for histechnical assistance.

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peptidases play an important role in cataractogenesis: an immunohistochemical study on lenses...

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