Mca-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH2

To investigate differences between activities related to phagocytosis in iris pigment epithelial (IPE) and retinal pigment epithelial (RPE) cells, an aspartic protease, cathepsin D (cat D), and a cysteine protease, cathepsin S (cat S), of IPE and RPE were studied. IPE and RPE cells were isolated from Long Evans rat eyes. The origin of the isolated cells was determined by pigmentation and cytokeratin labelling. The mRNA expressions of cat D and cat S in cultured IPE or RPE cells were investigated by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Enzyme activities of cat D and cat S in IPE or RPE cells were measured by using specific fluorogenic substrates, MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys-(Dnp)D-Arg-NH2 and Z-Val-Val-Arg-MCA, respectively. Western blot analysis of both proteins was also performed. The cultured cells, both of IPE and RPE cells were pigmented and showed positive labelling with an anti-cytokeratin monoclonal antibody. The cat D activity in RPE cells was 37 times that in IPE cells. The cat S activity in RPE cells was four times that in IPE cells. On the other hand, mRNA expression levels of cat D in RPE cells were at the same level with IPE cells, cat S mRNA expression in RPE cells were 10 times that in IPE cells. These results were also correlated with the Western blot analysis. In this study, we measured the characteristic expressions of cat D and S in IPE and RPE cells for the first time to compare their lysosomal activities. IPE cells have the lysosomal activities like RPE cells, however, the function of lysosomal activity in IPE cells is beneath RPE's. These results indicated that the ability of ROS digestion in IPE cells was not same as RPE cells.

Cathepsin E and cathepsin D are two major intracellular aspartic proteinases implicated in the physiological and pathological degradation of intra- and extracellular proteins. In this study, we designed and constructed highly sensitive synthetic decapeptide substrates for assays of cathepsins E and D based on the known sequence specificities of their cleavage sites. These substrates contain a highly fluorescent (7-methoxycoumarin-4-yl)acetyl (MOCAc) moiety and a quenching 2,4-dinitrophenyl (Dnp) group. When the Phe-Phe bond is cleaved, the fluorescence at an excitation wavelength of 328 nm and emission wavelength of 393 increases due to diminished quenching resulting from the separation of the fluorescent and quenching moieties. The first substrate, MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Le u-Lys(Dnp)gamma-NH2, in which the Lys-Pro combination at positions P5 and P4 was designed for specific interaction with cathepsin E, is hydrolyzed equally well by cathepsins E and D (kcat/Km = 10.9 microM(-1) x s(-1) for cathepsin E and 15.6 microM(-1) x s(-1) for cathepsin D). A very acidic pH optimum o was obtained for both enzymes. The second substrate, MOCAc-Gly-Lys-Pro-Ile-Ile-Phe-Phe-Arg-Le u-Lys(Dnp)gamma-NH2, in which the isoleucine residue at position P2 was meant to increase the specificity for cathepsin E, is also hydrolyzed equally by both enzymes (kcat/Km = 12.2 microM(-1) x s(-1) for cathepsin E and 16.3 microM(-1) x s(-1) for cathepsin D). The kcat/Km values for both substrates are greater than those for the best substrates for cathepsins E and D described so far. Unfortunately, each substrate shows little discrimination between cathepsin E and cathepsin D, suggesting that amino acids at positions far from the cleavage site are important for discrimination between the two enzymes. However, in combination with aspartic proteinase inhibitors, such as pepstatin A and Ascaris pepsin inhibitor, these substrates enable a rapid and sensitive determination of the precise levels of cathepsins E and D in crude cell extracts of various tissues and cells. Thus these substrates represent a potentially valuable tool for routine assays and for mechanistic studies on cathepsins E and D.

Mammalian sperm surface antigens are acquired either during spermatogenesis or sperm maturation in the epididymis. These antigens, many of which are hydrolytic enzymes, are actively synthesized and secreted by the resident epithelial cells and adsorbed to the sperm membrane as part of posttesticular sperm modification. In this study, we aimed to investigate the expression of cathepsin-D (CAT-D) in human reproductive tissues and its distribution on the sperm surface in different sperm conditions. Immunohistochemical results revealed the expression of CAT-D in the somatic Sertoli and Leydig cells without showing any immunoreactivity in any germ cells, despite their engagement of the acrosomal system. A strong immunoreactivity of anti-CAT-D was also detected in the epididymal epithelium, chiefly in the principal cells, which are known to actively synthesize and secrete proteins into the epididymal lumen. The absence of CAT-D in the clear cells was unexpected because these cells are known to engage the endosomal machinery. We further showed that CAT-D was anchored on the sperm surface confined to the postacrosomal region without any lateral redistribution within the membrane during sperm capacitation. However, the enzyme underwent changes to be an active form of a 29/30-kd doublet during sperm capacitation. Using CAT-D as a marker, we were able to demonstrate here localization of the enzyme in human reproductive tissues, as well as reveal membrane modification in human sperm during maturation and capacitation processes.