Degraded myelin-associated glycoprotein (dMAG) formation from pure human brain myelin-associated glycoprotein (MAG) is not mediated by calpain or cathepsin L-like activities

The myelin-associated glycoprotein (MAG) is a transmembrane cell adhesion molecule participating in myelin formation and maintenance. Calcium-activated/-dependent proteolysis of myelin-associated glycoprotein by calpain and cathepsin L-like activities has already been detected in purified myelin fractions, producing a soluble fragment, called degraded (d)MAG, characterized by the loss of the transmembrane and cytoplasmic domains. Here, we demonstrate and analyze dMAG formation from pure human brain myelin-associated glycoprotein. The activity never exhibited the high rate previously reported in human myelin fractions. Degradation is time-, temperature-, buffer- and structure-dependent, is inhibited at 4 degrees C and by denaturation of the sample, and is mediated by a trans-acting factor. There is no strict pH dependency of the proteolysis. Degradation was inhibited by excess aprotinin, but not by 1-10 micro g/mL aprotinin and was not eliminated by the use of an aprotinin-sepharose matrix during the purification. dMAG formation was not enhanced by calcium, nor inhibited by a wide variety of protease inhibitors, including specific calpain and cathepsin L inhibitors. Therefore, while cysteine proteases may be present in human myelin membrane fractions, they are not involved in dMAG formation from highly purified human brain myelin-associated glycoprotein preparations.     

Effects of Trypsin and Plasmin Treatment of Myelin on the Myelin-Associated Glycoprotein and Basic Protein

Human and rat myelin preparations were incubated with varying concentrations of trypsin and plasmin to determine the effects of these proteolytic enzymes on myelin-associated glycoprotein (MAG), basic protein, and other myelin proteins and to compare the effects with those of the neutral protease that was reported to be endogenous in myelin. Basic protein was most susceptible to degradation by both trypsin and plasmin, whereas MAG was relatively resistant to their actions. Under the assay conditions used, the highest concentrations of trypsin and plasmin degraded greater than 80% of the basic protein but less than 30% of the MAG, and lower concentrations caused significant loss of basic protein without appreciably affecting MAG. Neither trypsin nor plasmin caused a specific cleavage of MAG to a derivative of MAG (dMAG) in a manner analogous to the endogenous neutral protease. Thus the endogenous protease appears unique in converting human MAG to dMAG much more rapidly than it degrades basic protein. MAG is slowly degraded along with other proteins when myelin is treated with trypsin or plasmin, but it is less susceptible to their action than is basic protein.     

Susceptibility of the Myelin-Associated Glycoprotein and Basic Protein to a Neutral Protease in Highly Purified Myelin from Human and Rat Brain

Abstract: Incubation of highly purified human myelin at 25° and pH 8 in ammonium bicarbonate buffer resulted in the conversion of the myelin-associated glycoprotein (MAG) to a smaller derivative (dMAG) with an apparent molecular weight about 10,000 less. dMAG was stable and was not degraded to lower-molecular-weight breakdown products. Incubation of myelin under these conditions also resulted in the degradation of basic protein, but at a much slower rate. Half of the MAG was converted to dMAG in about 30 min, whereas degradation of half of the basic protein required 18 h of incubation. There was no significant loss of proteolipid, the Wolfgram doublet, or other myelin proteins during incubation for up to 18 h under these conditions. The formation of dMAG and the degradation of basic protein appear to be mediated by similar enzymatic activities; both processes exhibited broad pH optima in the neutral range, were prevented by briefly heating the myelin to 70° before incubation, and were stimulated by ammonium bicarbonate and other salts. Incubation of purified rat myelin also resulted in the formation of dMAG and the degradation of basic protein, but the conversion to dMAG occurred much more slowly than in human myelin preparations. In the rat, the percentage decreases in intact MAG and in basic protein were similar to each other and proceeded at rates comparable to the loss of basic protein in human myelin. These studies confirm and extend earlier demonstrations of neutral protease activity in purified myelin, and show that cleavage of MAG is one of the effects of this activity. The proteolytic activity affecting MAG and basic protein was not significantly reduced by further purification of the myelin on sucrose or CsCl gradients, suggesting that the neutral protease may be a myelin-related enzyme. The very high susceptibility of human MAG to this enzyme indicates that the effect of neutral protease on this glycoprotein should be considered in connection with demyelinating diseases.     

Conversion of myelin-associated glycoprotein (MAG) to a smaller derivative by calcium activated neutral protease (CANP)-like enzyme in myelin and inhibition by E-64 analogue

Using the immunoblot technique, we found that an incubation of purified human myelin in 10 mM Tris-HCl buffer at pH 7.5 resulted in the conversion of the myelinassociated glycoprotein (MAG) to a smaller derivative (dMAG). Exogenously added 5 mM CaCl₂ accelerated the conversion of MAG. In buffer containing more than 100 M of EGTA, the conversion was inhibited. In addition, the existence of endogenous calcium in purified myelin was confirmed using atomic absorption spectroscopy. The conversion was also inhibited partially by one of the thiol protease inhibitors, E-64 analogue (E-64-a). These observations suggest that the conversion of MAG is mediated by calcium-activated neutral protease (CANP)-like enzyme.     

Leishmania aethiopica: identification and characterization of cathepsin L-like cysteine protease genes

There is limited information on the biology and pathogenesis of Leishmania aethiopica, causative agent of cutaneous leishmaniasis (CL) in Ethiopia. In this study we have identified and characterized two cathepsin L-like cysteine protease genes, Laecpa and Laecpb, from L. aethiopica. The predicted amino acid sequence of Laecpa and Laecpb is more than 75% identical with homologous cathepsin L-like cysteine protease genes of other Leishmania species and less than 50% identical with human cathepsin L. Laecpa is expressed predominantly in the stationary, and to a lower level, during the amastigote stage while Laecpb is specifically expressed in the stationary stage of L. aethiopica development. Phylogenetic analysis showed that the two genes are grouped into separate clades which are the result of gene duplication. The isolation of these genes will be useful in developing Leishmania species specific diagnostics for molecular epidemiological studies and serves as a first step to study the role of cysteine proteases in L. aethiopica pathogenesis.     

Full-length cDNA of human cathepsin F predicts the presence of a cystatin domain at the N-terminus of the cysteine protease zymogen

A novel human cDNA encoding a cysteine protease of the papain family named cathepsin F is reported. The mature part of the predicted protease precursor displays between 26% and 42% identity to other human cysteine proteases while the proregion is unique by means of length and sequence. The very long proregion of the cathepsin F precursor (251 amino acid residues) can be divided into three regions: a C-terminal domain similar to the pro-segment of cathepsin L-like enzymes, a 50 residue flexible linker peptide, and an N-terminal domain predicted to adopt a cystatin-like fold. Cathepsin F would therefore be the first cysteine protease zymogen containing a cystatin-like domain.     

The human cysteine protease cathepsin V can compensate for murine cathepsin L in mouse epidermis and hair follicles

Mice lacking the ubiquitously expressed lysosomal cysteine protease cathepsin L, show a complex skin phenotype consisting of periodic hair loss and epidermal hyperplasia with hyperproliferation of basal epidermal keratinocytes, acanthosis and hyperkeratosis. The recently identified human cathepsin L-like enzyme cathepsin V, which is also termed cathepsin L2, is specifically expressed in cornea, testis, thymus, and epidermis. To date, in mice no cathepsin V orthologue with this typical expression pattern has been identified. Since cathepsin V has about 75% protein sequence identity to murine cathepsin L, we hypothesized that transgenic, keratinocyte-specific expression of cathepsin V in cathepsin L knockout mice might rescue the skin and hair phenotype. Thus, we generated a transgenic mouse line expressing cathepsin V under the control of the human keratin 14 promoter, which mimics the genuine cathepsin V expression pattern in human skin, by directing it to basal epidermal keratinocytes and the outer root sheath of hair follicles. Subsequently, transgenic mice were crossed with congenic cathepsin L knockout animals. The resulting mice show normalization of epidermal proliferation and normal epidermal thickness as well as rescue of the hair phenotype. These findings provide evidence for keratinocyte-specific pivotal functions of cathepsin L-like proteolytic activities in maintenance of epidermis and hair follicles and suggest, that cathepsin V may perform similar functions in human skin.     

Structural and functional conservation profiles of novel cathepsin L-like proteins identified in the Drosophila melanogaster genome

Cathepsin L is a cysteine protease which degrades connective tissue proteins including collagen, elastin, and fibronectin. In this study, five well-characterized cathepsin L proteins from different arthropods were used as query sequences for the Drosophila genome database. The search yielded 10 cathepsin L-like sequences, of which eight putatively represent novel cathepsin L-like proteins. To understand the phylogenetic relationship among these cathepsin L-like proteins, a phylogenetic tree was constructed based on their sequences. In addition, models of the tertiary structures of cathepsin L were constructed using homology modeling methods and subjected to molecular dynamics simulations to obtain reasonable structure to understand its dynamical behavior. Our findings demonstrate that all of the potential Drosophila cathepsin L-like proteins contain at least one cathepsin propeptide inhibitor domain. Multiple sequence alignment and homology models clearly highlight the conservation of active site residues, disulfide bonds, and amino acid residues critical for inhibitor binding. Furthermore, comparative modeling indicates that the sequence/structure/function profiles and active site architectures are conserved.     

Partial characterization of a novel cathepsin L-like protease from Fasciola hepatica

A 30-kDa protease, purified previously from Fasciola hepatica, was sequenced and the first 15 N-terminal residues were found to be 100% homologous to a region in the protein Fcp1c, which was cloned and expressed from F. hepatica. This terminal region was also 53 and 54% identical to two other cathepsin L-like proteases isolated from the same source. The 30-kDa protease demonstrated a specificity different from humancathepsin L when assayed with novel peptidyl enediones of the type Z-Phe-Ala-CH&dbond;CH(2)-CO(2)R (where R = Me/Et/Bu(t)). The ethyl ester peptide was a more efficient inhibitor of the protease than the corresponding methyl ester. This is in contrast to bovine cathepsin B and human cathepsin L where both are more readily inhibited by the methyl, rather than the ethyl ester peptide. These differences in the inhibition of the novel parasite protease may allow it to be exploited as a chemotherapeutic target.     

Isolation and molecular characterization of cathepsin L-like cysteine protease cDNAs from western flower thrips (Frankliniella occidentalis)

Cysteine proteases are predominant in thrips guts (TGs) and, therefore, a suitable target for selecting effective protease inhibitors against western flower thrips (Frankliniella occidentalis). We report the isolation of four full-length cysteine protease cDNA clones from thrips in a two-step PCR approach with degenerate oligonucleotides designed on conserved cathepsin L domains. At the deduced amino acid level, the clones possessed all functional and structural characteristics of cathepsin L, and showed high mutual identity and strong similarity with cathepsin L-like cysteine proteases from other insects and arthropods. Southern analysis indicated that a family of four closely related and 10-12 less-related genes encode the cathepsin L-like cysteine proteases in the thrips genome. Partial sequencing of genomic DNA demonstrated the presence of three introns in the coding DNA.     

Myelin instability and oligodendrocyte metabolism in myelin-deficient mutant mice

During the active phase of myelination in myelin-deficient mutant mice (mld), myelin basic protein (MBP) synthesis is defective and the myelin lamellae are uncompacted. In these mutants, we found a fast metabolism of the myelin-associated glycoprotein (MAG) and of sulfatides, and the presence of cholesterol esters and a degradation product of MAG, dMAG, indicating that mld myelin was unstable. The increased synthesis of MAG and Wolfgram protein, two proteins present in uncompacted myelin sheath and paranodal loops, was demonstrated by high levels of messengers. Simultaneously, we found an accumulation of inclusion bodies, vacuoles, and rough endoplasmic reticulum in mld oligodendrocytes. This material was heavily immunostained for MAG. Furthermore, the developmental change between the two molecular forms of MAG (p72MAG/p67MAG) was delayed in mld mice. In 85-d-old mld mice, the MBP content increased and myelin lamellae became better compacted. In these mutants, dMAG was absent and MAG mRNAs were found in normal amounts. Furthermore, the fine structure of mld oligodendrocytes was normal and the MAG immunostaining was similar to age-matched controls. These results support a functional role for MBP in maintaining the metabolic stability and the compact structure of myelin. Furthermore, in the absence of MBP and myelin compaction, the regulation of the synthesis of at least two membrane proteins related to myelin cannot proceed.     

Identification and characterization of the interactive proteins with cytotoxic T-lymphocyte antigen-2α

Cytotoxic T-lymphocyte antigen-2α (CTLA-2α) is a potent inhibitor of cathepsin L-like cysteine proteases. Recombinant CTLA-2α is known to be a potent, competitive inhibitor of cathepsin L-like cysteine proteases. In this study, cathepsin L, cathepsin C, and tubulointerstitial nephritis antigen-related protein 1 (TINAGL1) were identified as novel interactive proteins of CTLA-2α by the yeast two-hybrid screening system. The direct interactions and co-localization of these proteins with CTLA-2α were confirmed using co-immunoprecipitation and immunofluorescence staining, respectively. The disulfide-bonded CTLA-2α/cathepsin L complex was isolated from mouse tissue. CTLA-2α was found to be specific and consistently expressed on the maternal side of the mouse placenta. Double immunofluorescence analysis showed that CTLA-2α was co-localized with cathepsin L, cathepsin C, and TINAGL1 in placenta. A simple cell-based fluorescence assay revealed that CTLA-2α exhibited inhibitory activity toward cathepsin C in live cells, which indicated that CTLA-2α is a novel endogenous inhibitor of cathepsin C.     

Selective inhibition of the collagenolytic activity of human cathepsin K by altering its S2 subsite specificity

The primary specificity of papain-like cysteine proteases (family C1, clan CA) is determined by S2-P2 interactions. Despite the high amino acid sequence identities and structural similarities between cathepsins K and L, only cathepsin K is capable of cleaving interstitial collagens in their triple helical domains. To investigate this specificity, we have engineered the S2 pocket of human cathepsin K into a cathepsin L-like subsite. Using combinatorial fluorogenic substrate libraries, the P1-P4 substrate specificity of the cathepsin K variant, Tyr67Leu/Leu205Ala, was determined and compared with those of cathepsins K and L. The introduction of the double mutation into the S2 subsite of cathepsin K rendered the unique S2 binding preference of the protease for proline and leucine residues into a cathepsin L-like preference for bulky aromatic residues. Homology modeling and docking calculations supported the experimental findings. The cathepsin L-like S2 specificity of the mutant protein and the integrity of its catalytic site were confirmed by kinetic analysis of synthetic di- and tripeptide substrates as well as pH stability and pH activity profile studies. The loss of the ability to accept proline in the S2 binding pocket by the mutant protease completely abolished the collagenolytic activity of cathepsin K whereas its overall gelatinolytic activity remained unaffected. These results indicate that Tyr67 and Leu205 play a key role in the binding of proline residues in the S2 pocket of cathepsin K and are required for its unique collagenase activity.     

动物组织蛋白酶L=like基因家族的进化分析

目的：组织蛋白酶L-like家族是在溶酶体中发现的一类非常重要的半胱氨酸组织蛋白酶。其主要功能为催化各种蛋白质的水解,并通过水解蛋白质参与到许多的生理调节过程当中。根据序列比对分析和传统的功能分类,在动物中,组织蛋白酶L．1ike家族成员包括组织蛋白酶L、V、S、K、H和F。但是这些家族成员之间的进化关系仍然没有详细研究分析清楚。本课题主要研究组织蛋白酶L-like家族成员之间的进化关系。方法：本研究通过搜集整理22个物种的177条组织蛋白酶L-1ike家族蛋白的序列,并构建系统发育进化树来分析组织蛋白酶L-like家族各成员之间的进化关系。结果：序列数据结果显示,串联重复在组织蛋白酶L-1ike家族的进化过程中发生。斑马鱼的组织蛋白酶L,爪蟾的组织蛋白酶S和K,大鼠和小鼠的组织蛋白酶L都发生过明显的串联重复事件。进化树结果显示了组织蛋白酶H、S和K、L和V之间的进化关系,组织蛋白酶S和K在脊椎动物出现的进化过程中,从组织蛋白酶L中分化出来,与他们在脊椎动物体内的特异性功能,以及脊椎动物在进化过程中分化产生的特异性功能相对应。结论：在物种进化的过程中,组织蛋白酶L-1ike家族成员F、H、S和K、L和V按时间顺序分化,这表明组织蛋白酶L-1ike基因家族结构和功能的分化与新的物种和新的功能出现密切相关。     

Site-directed mutagenesis, proteolytic cleavage, and activation of human proheparanase

Heparanase is an endo-beta-D-glucuronidase that degrades heparan sulfate in the extracellular matrix and cell surfaces. Human proheparanase is produced as a latent 65-kDa polypeptide undergoing processing at two potential proteolytic cleavage sites, located at Glu109-Ser110 (site 1) and Gln157-Lys158 (site 2). Cleavage of proheparanase yields 8- and 50-kDa subunits that heterodimerize to form the active enzyme. The fate of the linker segment (Ser110-Gln157) residing between the two subunits, the mode of processing, and the protease(s) engaged in proheparanase processing are currently unknown. We applied multiple site-directed mutagenesis and deletions to study the nature of the potential cleavage sites and amino acids essential for processing of proheparanase in transfected human choriocarcinoma cells devoid of endogenous heparanase but possessing the enzymatic machinery for proper processing and activation of the proenzyme. Although mutagenesis at site 1 and its flanking sequences failed to identify critical residues for proteolytic cleavage, processing at site 2 required a bulky hydrophobic amino acid at position 156 (i.e. P2 of the cleavage site). Substitution of Tyr156 by Ala or Glu, but not Val, resulted in cleavage at an upstream site in the linker segment, yielding an improperly processed inactive enzyme. Processing of the latent 65-kDa proheparanase in transfected Jar cells was inhibited by a cell-permeable inhibitor of cathepsin L. Moreover, recombinant 65-kDa proheparanase was processed and activated by cathepsin L in a cell-free system. Altogether, these results suggest that proheparanase processing at site 2 is brought about by cathepsin L-like proteases. The involvement of other members of the cathepsin family with specificity to bulky hydrophobic residues cannot be excluded. Our results and a three-dimensional model of the enzyme are expected to accelerate the design of inhibitory molecules capable of suppressing heparanase-mediated enhancement of tumor angiogenesis and metastasis.     

Developmental alterations in molecular weights of proteins in the human central nervous system that react with antibodies against myelin- associated glycoprotein

By the use of a rat IgG monoclonal antibody (mab), a mouse mab and human serum containing an IgM mab, all of which react with isolated human myelin-associated glycoprotein (MAG) on immunoblots and bind only to proteins with relative mobilities identical to MAG and dMAG on immunoblots of homogenates of adult human spinal cord, we demonstrated the following: in homogenates of central nervous system tissue from human fetuses of gestational ages that antedate myelination, the anti- MAG antibodies react only with proteins with molecular weights of 250,000 or larger. During myelination the molecular weights of proteins with which the anti-MAG antibodies react shift towards the lower molecular weights found in adult myelin. Amongst those central nervous system regions examined, the shift towards the low molecular weights occurred earliest in the region that is first to become myelinated and latest in the one that is the last to myelinate. Once myelination is completed, the antibodies react only with proteins with relative mobilities identical to those of MAG and dMAG. These developmental changes in molecular weights of "MAG-related proteins" may prove useful as an index of chemical processes on the basis of which myelination occurs.     

Novel cathepsin L-like protease from dermestid beetle Dermestes frischii maggot

A novel cathepsin L-like protease from dermestid beetle Dermestes frischii maggot guts was obtained and investigated. The protease was isolated through affinity chromatography at arginine-diasorb followed by FPLC gel-filtration at Superdex 75. Protease is active against chromogenic peptide substrates, containing Arg or Leu in P1 position and a hydrophobic residue in P2 position. PH optimum is about 4,5 and temperature optimum at 40 °C. Enzyme is inhibited completely by HgCl₂ and leupeptin that prove it’s belonging to cysteine proteases of papain family.cDNA analysis of cathepsin L-like protease showed that protein sequence consists of 339 amino acid residues. Mature cysteine protease contains 219 amino acid residues corresponding to molecular mass 24027.20 Da. Residues of the active site were identified: Gln¹⁴⁰, Cys¹⁴⁶, His²⁸⁵, Asn³⁰⁶ and Trp³⁰⁸. Calculated pI is 4,73. The amino acid sequence of the cystein protease from dermestid beetle displays high structural homology with cathepsin L of other insects.     

Human cathepsin V functional expression, tissue distribution, electrostatic surface potential, enzymatic characterization, and chromosomal localization

Cathepsin V, a thymus and testis-specific human cysteine protease, was expressed in Pichia pastoris, and its physicokinetic properties were determined. Recombinant procathepsin V is autocatalytically activated at acidic pH and is effectively inhibited by various cysteine protease class-specific inhibitors. The S2P2 subsite specificity of cathepsin V was found to be intermediate between those of cathepsins S and L. The substrate binding pocket, S2, accepted both aromatic and nonaromatic hydrophobic residues, whereas cathepsins L and S preferred either an aromatic or nonaromatic hydrophobic residue, respectively. In contrast to cathepsin L, but similar to cathepsin S, cathepsin V exhibited only a very weak collagenolytic activity. Furthermore, cathepsin V was determined to be significantly more stable at mildly acidic and neutral pH than cathepsin L, but distinctly less stable than cathepsin S. A homology structure model of cathepsin V revealed completely different electrostatic potentials on the molecular surface when compared with human cathepsin L. The model-based electrostatic potential of human cathepsin V was neutral to weakly positive at and in the vicinity of the active site cleft, whereas that of cathepsin L was negative over extended regions of the surface. Surprisingly, the electrostatic potential of the human cathepsin V model structure resembled that of the model structure of mouse cathepsin L. These differences in the electrostatic potential at the molecular surfaces provide a reactivity determinant that may be the source of differences in substrate selectivity and pH stability. Cathepsin V was mapped to the chromosomal region 9q22.2, a site adjacent to the cathepsin L locus. The high sequence identity and the overlapping chromosomal gene loci suggest that both proteases evolved from an ancestral cathepsin L-like precursor by gene duplication.