Serine protease which exhibits a preference for Arg over Lys in the substrate P1 position and for Ser or Pro in the P2 position. Shows activity against amyloid precursor protein, myelin basic protein, gelatin, casein and extracellular matrix proteins such as fibronectin, laminin, vitronectin and collagen. Degrades alpha-synuclein and prevents its polymerization, indicating that it may be involved in the pathogenesis of Parkinson disease and other synucleinopathies. May be involved in regulation of axon outgrowth following spinal cord injury. Tumor cells treated with a neutralizing KLK6 antibody migrate less than control cells, suggesting a role in invasion and metastasis.
Human kallikrein 6 (hK6), a trypsin-like serine protease, is a newly identified member of the kallikrein gene family. Its involvement in inflammatory CNS lesions and in demyelination has been reported. Recent work has suggested that expression of this enzyme is significantly elevated in patients with ovarian cancer. We have identified many tumour cell lines that secrete hK6, but its physiological role is unknown. Here, we try to unveil the role of this kallikrein in the metastasis and invasion of tumour cells. We demonstrate that purified human recombinant hK6 can cleave gelatin in zymography and can efficiently degrade high-molecular-weight extracellular matrix proteins such as fibronectin, laminin, vitronectin and collagen. In Boyden chamber assays, we found that tumour cells treated with a neutralizing hK6 antibody migrate less than control cells. We conclude that hK6 might play a role in the invasion and metastasis of tumour cells and may be a candidate therapeutic target.
The human kallikreins are a large multigene family of closely related serine-type proteases. In this regard, they are similar to the multigene kallikrein families characterized in mice and rats. There is a much more extensive body of knowledge regarding the function of mouse and rat kallikreins in comparison with the human kallikreins. Human kallikrein 6 has been proposed as the homologue to rat myelencephalon-specific protease, an arginine-specific degradative-type protease abundantly expressed in the central nervous system and implicated in demyelinating disease. We present the x-ray crystal structure of mature, active recombinant human kallikrein 6 at 1.75-A resolution. This high resolution model provides the first three-dimensional view of one of the human kallikreins and one of only a few structures of serine proteases predominantly expressed in the central nervous system. Enzymatic data are presented that support the identification of human kallikrein 6 as the functional homologue of rat myelencephalon-specific protease and are corroborated by a molecular phylogenetic analysis. Furthermore, the x-ray data provide support for the characterization of human kallikrein 6 as a degradative protease with structural features more similar to trypsin than the regulatory kallikreins.
Human kallikrein 6 (hK6) is abundantly expressed in the central nervous system and is implicated in demyelinating disease. This study provided biochemical data about the substrate specificity and activation of hK6 by glycosaminoglycans and by kosmotropic salts, which followed the Hofmeister series. The screening of fluorescence resonance energy transfer (FRET) peptide families derived from Abz-KLRSSKQ-EDDnp resulted in the finding that Abz-AFRFSQ-EDDnp (where Abz is ortho-aminobenzoic acid and EDDnp is N-[2,4-dinitrophenyl]ethylenediamine)) is the best synthetic substrate described so far for hK6 (kcat/Km 38,667 s(-1) mm(-1)). It is noteworthy that the AFRFS sequence was found as a motif in the amino-terminal domain of seven human ionotropic glutamate receptor subunits. We also examined the hK6 hydrolytic activity on FRET peptides derived from human myelin basic protein, precursor of the Abeta amyloid peptide, reactive center loop of alpha1-antichymotrypsin, plasminogen, and maturation and inactivation cleavage sites of hK6, which were described earlier as natural substrates for hK6. The best substrates were derived from myelin basic protein. The hK6 maturation cleavage site was poorly hydrolyzed, and no evidence was found to support a two-step self-activation process reported previously. Finally, we assayed FRET peptides derived from sequences that span the cleavage sites for activation of protease-activated receptors (PAR) 1-4, and only the substrate with the PAR 2 sequence was hydrolyzed. These results further supported the hypothesis that hK6 expressed in the central nervous system is involved in normal myelin turnover/demyelination processes, but it is unlikely to self-activate. This report also suggested the possible modulation of ionotropic glutamate receptors and activation of PAR 2 by hK6.
Kallikrein 6 (K6) is a member of the kallikrein gene family that comprises 15 structurally and functionally related serine proteases. In prior studies we showed that, while this trypsin-like enzyme is preferentially expressed in neurons and oligodendroglia of the adult central nervous system (CNS), it is up-regulated at sites of injury due to expression by infiltrating immune and resident CNS cells. Given this background we hypothesized that K6 is a key contributor to the pathophysiology of traumatic spinal cord injury (SCI), influencing neural repair and regeneration. Examination of K6 expression following contusion injury to the adult rat cord, and in cases of human traumatic SCI, indicated significant elevations at acute and chronic time points, not only at the injury site but also in cord segments above and below. Elevations in K6 were particularly prominent in macrophages, microglia and reactive astrocytes. To determine potential effects of elevated K6 on the regeneration environment, the ability of neurons to adhere to and extend processes on substrata which had been exposed to recombinant K6 was examined. Limited (1 h) or excess (24 h) K6-mediated proteolytic digestion of a growth-facilitatory substrate, laminin, significantly decreased neurite outgrowth. By contrast, similar hydrolysis of a growth-inhibitory substrate, aggrecan, significantly increased neurite extension and cell adherence. These data support the hypothesis that K6 enzymatic cascades mediate events secondary to spinal cord trauma, including dynamic modification of the capacity for axon outgrowth.
Accumulation of insoluble alpha-synuclein aggregates in the brain is characteristic of Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Although numerous studies on the aggregation properties of alpha-synuclein have been reported, little is known about its degradation so far. In view of proteolytic degradation, we have found that the serine protease neurosin (kallikrein-6) degrades alpha-synuclein and co-localizes with pathological inclusions such as Lewy bodies and glial cytoplasmic inclusions. In vitro study showed that neurosin prevented alpha-synuclein polymerization by reducing the amount of monomer and also by generating fragmented alpha-synucleins that themselves inhibited the polymerization. Upon cellular stress, neurosin was released from mitochondria to the cytosol, which resulted in the increase of degraded alpha-synuclein species. Down-regulation of neurosin caused accumulation of alpha-synuclein within cultured cells. Thus we concluded that neurosin plays a significant role in physiological alpha-synuclein degradation and also in the pathogenesis of synucleinopathies.
Human kallikrein 6 (hK6) is a trypsin-like serine protease, member of the human kallikrein gene family. Studies suggested a potential involvement of hK6 in the development and progression of Alzheimer's disease. The serum levels of hK6 might be used as a biomarker for ovarian cancer. To gain insights into the physiological role of this enzyme, we sought to determine its substrate specificity and its interactions with various inhibitors. We produced the proform of hK6 and showed that this enzyme was able to autoactivate, as well as proteolyse itself, leading to inactivation. Kinetic studies indicated that hK6 cleaved with much higher efficiency after Arg than Lys and with a preference for Ser or Pro in the P2 position. The efficient degradation of fibrinogen and collagen types I and IV by hK6 indicated that this kallikrein might play a role in tissue remodeling and/or tumor invasion and metastasis. We also demonstrated proteolysis of amyloid precursor protein by hK6 and determined the cleavage sites at the N-terminal end of the protein. Inhibition of hK6 was achieved via binding to different serpins, among which antithrombin III was the most efficient.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionUniProtKB
BACKGROUND: Human kallikrein 6 (hK6) is significantly increased in serum in many patients with ovarian cancer and may have a role in amyloid precursor processing and Alzheimer disease. The forms of hK6 in biological fluids are poorly characterized. METHODS: hK6 protein was immunoaffinity-purified and positively identified by Western blotting, N-terminal sequencing, and mass spectrometry. hK6 in cerebrospinal fluid (CSF), milk, ascites, and serum was size-fractionated by chromatography and then measured by a highly sensitive and specific immunoassay. Hybrid assays were performed to detect the possible interactions between hK6 and proteinase inhibitors in CSF, milk, ascites fluid, and serum. RESULTS: N-Terminal sequencing identified hK6 in the proform in both CSF and milk. hK6 exists in two forms in milk and ascites fluid: a free form with a molecular mass of approximately 25 kDa and a higher molecular mass form. Hybrid sandwich assays (capture antibody for hK6 and detection antibody for inhibitors), utilizing a panel of known serine protease inhibitors, indicated that alpha(1)-antichymotrypsin forms a complex with hK6 in milk and ascites fluid. Only the free form of hK6 was detected in CSF and serum. CONCLUSIONS: hK6 exists mainly as a proenzyme in milk and CSF. A fraction of this enzyme is partially complexed with alpha(1)-antichymotrypsin in milk and ascites fluid of ovarian cancer patients.
Evidence
2:
Inferred from Physical InteractionUniProtKB
Human kallikrein 6 (hK6) is a trypsin-like serine protease, member of the human kallikrein gene family. Studies suggested a potential involvement of hK6 in the development and progression of Alzheimer's disease. The serum levels of hK6 might be used as a biomarker for ovarian cancer. To gain insights into the physiological role of this enzyme, we sought to determine its substrate specificity and its interactions with various inhibitors. We produced the proform of hK6 and showed that this enzyme was able to autoactivate, as well as proteolyse itself, leading to inactivation. Kinetic studies indicated that hK6 cleaved with much higher efficiency after Arg than Lys and with a preference for Ser or Pro in the P2 position. The efficient degradation of fibrinogen and collagen types I and IV by hK6 indicated that this kallikrein might play a role in tissue remodeling and/or tumor invasion and metastasis. We also demonstrated proteolysis of amyloid precursor protein by hK6 and determined the cleavage sites at the N-terminal end of the protein. Inhibition of hK6 was achieved via binding to different serpins, among which antithrombin III was the most efficient.
Catalysis of the hydrolysis of internal, alpha-peptide bonds in a polypeptide chain by a catalytic mechanism that involves a catalytic triad consisting of a serine nucleophile that is activated by a proton relay involving an acidic residue (e.g. aspartate or glutamate) and a basic residue (usually histidine).
Serine proteinases like thrombin can signal to cells by the cleavage/activation of proteinase-activated receptors (PARs). Although thrombin is a recognized physiological activator of PAR(1) and PAR(4), the endogenous enzymes responsible for activating PAR(2) in settings other than the gastrointestinal system, where trypsin can activate PAR(2), are unknown. We tested the hypothesis that the human tissue kallikrein (hK) family of proteinases regulates PAR signaling by using the following: 1) a high pressure liquid chromatography (HPLC)-mass spectral analysis of the cleavage products yielded upon incubation of hK5, -6, and -14 with synthetic PAR N-terminal peptide sequences representing the cleavage/activation motifs of PAR(1), PAR(2), and PAR(4); 2) PAR-dependent calcium signaling responses in cells expressing PAR(1), PAR(2), and PAR(4) and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR(4)-dependent rat and human platelet aggregation assay. We found that hK5, -6, and -14 all yielded PAR peptide cleavage sequences consistent with either receptor activation or inactivation/disarming. Furthermore, hK14 was able to activate PAR(1), PAR(2), and PAR(4) and to disarm/inhibit PAR(1). Although hK5 and -6 were also able to activate PAR(2), they failed to cause PAR(4)-dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR(2)-mediated calcium signaling differed. Our data indicate that in physiological settings, hKs may represent important endogenous regulators of the PARs and that different hKs can have differential actions on PAR(1), PAR(2), and PAR(4).
Human kallikrein 6 (hK6) is a trypsin-like serine protease, member of the human kallikrein gene family. Studies suggested a potential involvement of hK6 in the development and progression of Alzheimer's disease. The serum levels of hK6 might be used as a biomarker for ovarian cancer. To gain insights into the physiological role of this enzyme, we sought to determine its substrate specificity and its interactions with various inhibitors. We produced the proform of hK6 and showed that this enzyme was able to autoactivate, as well as proteolyse itself, leading to inactivation. Kinetic studies indicated that hK6 cleaved with much higher efficiency after Arg than Lys and with a preference for Ser or Pro in the P2 position. The efficient degradation of fibrinogen and collagen types I and IV by hK6 indicated that this kallikrein might play a role in tissue remodeling and/or tumor invasion and metastasis. We also demonstrated proteolysis of amyloid precursor protein by hK6 and determined the cleavage sites at the N-terminal end of the protein. Inhibition of hK6 was achieved via binding to different serpins, among which antithrombin III was the most efficient.
The chemical reactions and pathways involving amyloid precursor protein (APP), the precursor of beta-amyloid, a glycoprotein associated with Alzheimer's disease.
BACKGROUND: Human kallikrein 6 (hK6) is significantly increased in serum in many patients with ovarian cancer and may have a role in amyloid precursor processing and Alzheimer disease. The forms of hK6 in biological fluids are poorly characterized. METHODS: hK6 protein was immunoaffinity-purified and positively identified by Western blotting, N-terminal sequencing, and mass spectrometry. hK6 in cerebrospinal fluid (CSF), milk, ascites, and serum was size-fractionated by chromatography and then measured by a highly sensitive and specific immunoassay. Hybrid assays were performed to detect the possible interactions between hK6 and proteinase inhibitors in CSF, milk, ascites fluid, and serum. RESULTS: N-Terminal sequencing identified hK6 in the proform in both CSF and milk. hK6 exists in two forms in milk and ascites fluid: a free form with a molecular mass of approximately 25 kDa and a higher molecular mass form. Hybrid sandwich assays (capture antibody for hK6 and detection antibody for inhibitors), utilizing a panel of known serine protease inhibitors, indicated that alpha(1)-antichymotrypsin forms a complex with hK6 in milk and ascites fluid. Only the free form of hK6 was detected in CSF and serum. CONCLUSIONS: hK6 exists mainly as a proenzyme in milk and CSF. A fraction of this enzyme is partially complexed with alpha(1)-antichymotrypsin in milk and ascites fluid of ovarian cancer patients.
The process whose specific outcome is the progression of the central nervous system over time, from its formation to the mature structure. The central nervous system is the core nervous system that serves an integrating and coordinating function. In vertebrates it consists of the brain, spinal cord and spinal nerves. In those invertebrates with a central nervous system it typically consists of a brain, cerebral ganglia and a nerve cord.
Kallikreins are a subgroup of the serine protease family of enzymes. Until recently, it was thought that the human kallikrein gene family includes only three members. Over the past 3 years, the human kallikrein gene locus on chromosome 19q13.4 has been characterized. This family includes 15 members for which new nomenclature has been established. A number of kallikreins are expressed in the central nervous system (CNS). Experimental evidence has shown that at least two kallikreins, KLK6 and KLK8, have potential functions in the CNS. KLK8 (neuropsin) is highly expressed in brain tissues and may play a role in brain development, plasticity and response to stress. Of particular interest is the possible involvement of kallikreins in the pathogenesis of Alzheimer's disease (AD). KLK6 (zyme/protease M/neurosin) seems to be down regulated in serum and tissues of Alzheimer's disease patients and may be involved in amyloid metabolism.
The proteolytic chemical reactions and pathways resulting in the breakdown of collagen in the extracellular matrix, usually carried out by proteases secreted by nearby cells.
Human kallikrein 6 (hK6) is a trypsin-like serine protease, member of the human kallikrein gene family. Studies suggested a potential involvement of hK6 in the development and progression of Alzheimer's disease. The serum levels of hK6 might be used as a biomarker for ovarian cancer. To gain insights into the physiological role of this enzyme, we sought to determine its substrate specificity and its interactions with various inhibitors. We produced the proform of hK6 and showed that this enzyme was able to autoactivate, as well as proteolyse itself, leading to inactivation. Kinetic studies indicated that hK6 cleaved with much higher efficiency after Arg than Lys and with a preference for Ser or Pro in the P2 position. The efficient degradation of fibrinogen and collagen types I and IV by hK6 indicated that this kallikrein might play a role in tissue remodeling and/or tumor invasion and metastasis. We also demonstrated proteolysis of amyloid precursor protein by hK6 and determined the cleavage sites at the N-terminal end of the protein. Inhibition of hK6 was achieved via binding to different serpins, among which antithrombin III was the most efficient.
The chemical reactions and pathways involving any hormone, naturally occurring substances secreted by specialized cells that affects the metabolism or behavior of other cells possessing functional receptors for the hormone.
J. Histochem. Cytochem. 49, 1431-1441 (2001)[PubMed:11668196]
The KLK6 gene is a new member of the human kallikrein gene family and encodes for a secreted protease, human kallikrein 6 (hK6; also known as zyme/protease M/neurosin). No study has as yet reported detailed immunohistochemical localization of hK6 in human tissues. Our purpose was to examine the expression of hK6 in human tissues by immunohistochemistry. We have analyzed 199 paraffin blocks from archival, current, and autopsy material prepared from almost every normal human tissue. We employed an hK6-specific polyclonal rabbit antibody and avidin-biotin to localize hK6 by IHC. The staining pattern, the distribution of the immunostaining, and its intensity were studied in detail. The IHC expression of zyme was generally cytoplasmic. Various normal human tissues expressed the protein abundantly. Glandular epithelia constituted the main immunoexpression sites, with representative organs being the breast, prostate, kidney, endometrium, colon, appendix, salivary glands, bile ducts, and gallbladder. The small intestine, stomach, endocervix, Fallopian tube, epididymis, bronchus, and upper respiratory tract showed a focal expression as well. Choroid plexus epithelium, peripheral nerves, and some neuroendocrine cells (including the islets of Langerhans, cells in the anterior pituitary gland, and adrenal medulla) expressed the protein strongly and diffusely. A characteristic immunostaining was observed in the Hassall's corpuscles of the thymus, the oxyphilic cells of the thyroid and parathyroid glands, the primordial follicles of the ovary, dendritic cells mainly in the spleen, and in various cells of the placenta.
The process in which myelin sheaths are formed and maintained around neurons. Oligodendrocytes in the brain and spinal cord and Schwann cells in the peripheral nervous system wrap axons with compact layers of their plasma membrane. Adjacent myelin segments are separated by a non-myelinated stretch of axon called a node of Ranvier.
J. Histochem. Cytochem. 49, 1431-1441 (2001)[PubMed:11668196]
The KLK6 gene is a new member of the human kallikrein gene family and encodes for a secreted protease, human kallikrein 6 (hK6; also known as zyme/protease M/neurosin). No study has as yet reported detailed immunohistochemical localization of hK6 in human tissues. Our purpose was to examine the expression of hK6 in human tissues by immunohistochemistry. We have analyzed 199 paraffin blocks from archival, current, and autopsy material prepared from almost every normal human tissue. We employed an hK6-specific polyclonal rabbit antibody and avidin-biotin to localize hK6 by IHC. The staining pattern, the distribution of the immunostaining, and its intensity were studied in detail. The IHC expression of zyme was generally cytoplasmic. Various normal human tissues expressed the protein abundantly. Glandular epithelia constituted the main immunoexpression sites, with representative organs being the breast, prostate, kidney, endometrium, colon, appendix, salivary glands, bile ducts, and gallbladder. The small intestine, stomach, endocervix, Fallopian tube, epididymis, bronchus, and upper respiratory tract showed a focal expression as well. Choroid plexus epithelium, peripheral nerves, and some neuroendocrine cells (including the islets of Langerhans, cells in the anterior pituitary gland, and adrenal medulla) expressed the protein strongly and diffusely. A characteristic immunostaining was observed in the Hassall's corpuscles of the thymus, the oxyphilic cells of the thyroid and parathyroid glands, the primordial follicles of the ovary, dendritic cells mainly in the spleen, and in various cells of the placenta.
Serine proteinases like thrombin can signal to cells by the cleavage/activation of proteinase-activated receptors (PARs). Although thrombin is a recognized physiological activator of PAR(1) and PAR(4), the endogenous enzymes responsible for activating PAR(2) in settings other than the gastrointestinal system, where trypsin can activate PAR(2), are unknown. We tested the hypothesis that the human tissue kallikrein (hK) family of proteinases regulates PAR signaling by using the following: 1) a high pressure liquid chromatography (HPLC)-mass spectral analysis of the cleavage products yielded upon incubation of hK5, -6, and -14 with synthetic PAR N-terminal peptide sequences representing the cleavage/activation motifs of PAR(1), PAR(2), and PAR(4); 2) PAR-dependent calcium signaling responses in cells expressing PAR(1), PAR(2), and PAR(4) and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR(4)-dependent rat and human platelet aggregation assay. We found that hK5, -6, and -14 all yielded PAR peptide cleavage sequences consistent with either receptor activation or inactivation/disarming. Furthermore, hK14 was able to activate PAR(1), PAR(2), and PAR(4) and to disarm/inhibit PAR(1). Although hK5 and -6 were also able to activate PAR(2), they failed to cause PAR(4)-dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR(2)-mediated calcium signaling differed. Our data indicate that in physiological settings, hKs may represent important endogenous regulators of the PARs and that different hKs can have differential actions on PAR(1), PAR(2), and PAR(4).
Processing which a protein carries out itself. This involves actions such as the autolytic removal of residues to generate the mature form of the protein.
Human kallikrein 6 (hK6) is a trypsin-like serine protease, member of the human kallikrein gene family. Studies suggested a potential involvement of hK6 in the development and progression of Alzheimer's disease. The serum levels of hK6 might be used as a biomarker for ovarian cancer. To gain insights into the physiological role of this enzyme, we sought to determine its substrate specificity and its interactions with various inhibitors. We produced the proform of hK6 and showed that this enzyme was able to autoactivate, as well as proteolyse itself, leading to inactivation. Kinetic studies indicated that hK6 cleaved with much higher efficiency after Arg than Lys and with a preference for Ser or Pro in the P2 position. The efficient degradation of fibrinogen and collagen types I and IV by hK6 indicated that this kallikrein might play a role in tissue remodeling and/or tumor invasion and metastasis. We also demonstrated proteolysis of amyloid precursor protein by hK6 and determined the cleavage sites at the N-terminal end of the protein. Inhibition of hK6 was achieved via binding to different serpins, among which antithrombin III was the most efficient.
Any process that modulates the frequency, rate or extent of cell differentiation, the process in which relatively unspecialized cells acquire specialized structural and functional features.
J. Histochem. Cytochem. 49, 1431-1441 (2001)[PubMed:11668196]
The KLK6 gene is a new member of the human kallikrein gene family and encodes for a secreted protease, human kallikrein 6 (hK6; also known as zyme/protease M/neurosin). No study has as yet reported detailed immunohistochemical localization of hK6 in human tissues. Our purpose was to examine the expression of hK6 in human tissues by immunohistochemistry. We have analyzed 199 paraffin blocks from archival, current, and autopsy material prepared from almost every normal human tissue. We employed an hK6-specific polyclonal rabbit antibody and avidin-biotin to localize hK6 by IHC. The staining pattern, the distribution of the immunostaining, and its intensity were studied in detail. The IHC expression of zyme was generally cytoplasmic. Various normal human tissues expressed the protein abundantly. Glandular epithelia constituted the main immunoexpression sites, with representative organs being the breast, prostate, kidney, endometrium, colon, appendix, salivary glands, bile ducts, and gallbladder. The small intestine, stomach, endocervix, Fallopian tube, epididymis, bronchus, and upper respiratory tract showed a focal expression as well. Choroid plexus epithelium, peripheral nerves, and some neuroendocrine cells (including the islets of Langerhans, cells in the anterior pituitary gland, and adrenal medulla) expressed the protein strongly and diffusely. A characteristic immunostaining was observed in the Hassall's corpuscles of the thymus, the oxyphilic cells of the thyroid and parathyroid glands, the primordial follicles of the ovary, dendritic cells mainly in the spleen, and in various cells of the placenta.
Any process that modulates the rate, frequency or extent of neuron projection development. Neuron projection development is the process whose specific outcome is the progression of a neuron projection over time, from its formation to the mature structure. A neuron projection is any process extending from a neural cell, such as axons or dendrites (collectively called neurites).
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a stimulus indicating damage to the organism.
Zyme/protease M/neurosin/human kallikrein 6 (hK6) is a member of the human kallikrein family of trypsin-like serine proteinases and was originally identified as being down-regulated in metastatic breast and ovarian tumors when compared with corresponding primary tumors. Recent evidence suggests that hK6 may serve as a circulating tumor marker in ovarian cancers. In addition, it was described in the brain of Parkinson's disease and Alzheimer's disease patients, where it is implicated in amyloid precursor protein processing. It is thus a biomarker for these diseases. To examine the mechanism of activation of hK6, we have solved the structure of its proform, the first of a human kallikrein family member. The proenzyme displays a fold that exhibits chimeric features between those of trypsinogen and other family members. It lacks the characteristic "kallikrein loop" and forms the six disulfide bridges of trypsin. Pro-hK6 displays a completely closed specificity pocket and a unique conformation of the regions involved in structural rearrangements upon proteolytic cleavage activation. This points to a novel activation mechanism, which could be extrapolated to other human kallikreins.
Human kallikrein 6 (hK6) is a trypsin-like serine protease, member of the human kallikrein gene family. Studies suggested a potential involvement of hK6 in the development and progression of Alzheimer's disease. The serum levels of hK6 might be used as a biomarker for ovarian cancer. To gain insights into the physiological role of this enzyme, we sought to determine its substrate specificity and its interactions with various inhibitors. We produced the proform of hK6 and showed that this enzyme was able to autoactivate, as well as proteolyse itself, leading to inactivation. Kinetic studies indicated that hK6 cleaved with much higher efficiency after Arg than Lys and with a preference for Ser or Pro in the P2 position. The efficient degradation of fibrinogen and collagen types I and IV by hK6 indicated that this kallikrein might play a role in tissue remodeling and/or tumor invasion and metastasis. We also demonstrated proteolysis of amyloid precursor protein by hK6 and determined the cleavage sites at the N-terminal end of the protein. Inhibition of hK6 was achieved via binding to different serpins, among which antithrombin III was the most efficient.
Inhibited by a range of serine protease inhibitors including soybean trypsin inhibitor, benzamidine and serpins. Activated by a range of glycosaminoglycans including chondroitin sulfate, dermatan sulfate, heparan sulfate and heparin.
Human kallikrein 6 (hK6) is a trypsin-like serine protease, member of the human kallikrein gene family. Studies suggested a potential involvement of hK6 in the development and progression of Alzheimer's disease. The serum levels of hK6 might be used as a biomarker for ovarian cancer. To gain insights into the physiological role of this enzyme, we sought to determine its substrate specificity and its interactions with various inhibitors. We produced the proform of hK6 and showed that this enzyme was able to autoactivate, as well as proteolyse itself, leading to inactivation. Kinetic studies indicated that hK6 cleaved with much higher efficiency after Arg than Lys and with a preference for Ser or Pro in the P2 position. The efficient degradation of fibrinogen and collagen types I and IV by hK6 indicated that this kallikrein might play a role in tissue remodeling and/or tumor invasion and metastasis. We also demonstrated proteolysis of amyloid precursor protein by hK6 and determined the cleavage sites at the N-terminal end of the protein. Inhibition of hK6 was achieved via binding to different serpins, among which antithrombin III was the most efficient.
Human kallikrein 6 (hK6) is abundantly expressed in the central nervous system and is implicated in demyelinating disease. This study provided biochemical data about the substrate specificity and activation of hK6 by glycosaminoglycans and by kosmotropic salts, which followed the Hofmeister series. The screening of fluorescence resonance energy transfer (FRET) peptide families derived from Abz-KLRSSKQ-EDDnp resulted in the finding that Abz-AFRFSQ-EDDnp (where Abz is ortho-aminobenzoic acid and EDDnp is N-[2,4-dinitrophenyl]ethylenediamine)) is the best synthetic substrate described so far for hK6 (kcat/Km 38,667 s(-1) mm(-1)). It is noteworthy that the AFRFS sequence was found as a motif in the amino-terminal domain of seven human ionotropic glutamate receptor subunits. We also examined the hK6 hydrolytic activity on FRET peptides derived from human myelin basic protein, precursor of the Abeta amyloid peptide, reactive center loop of alpha1-antichymotrypsin, plasminogen, and maturation and inactivation cleavage sites of hK6, which were described earlier as natural substrates for hK6. The best substrates were derived from myelin basic protein. The hK6 maturation cleavage site was poorly hydrolyzed, and no evidence was found to support a two-step self-activation process reported previously. Finally, we assayed FRET peptides derived from sequences that span the cleavage sites for activation of protease-activated receptors (PAR) 1-4, and only the substrate with the PAR 2 sequence was hydrolyzed. These results further supported the hypothesis that hK6 expressed in the central nervous system is involved in normal myelin turnover/demyelination processes, but it is unlikely to self-activate. This report also suggested the possible modulation of ionotropic glutamate receptors and activation of PAR 2 by hK6.
Enzyme which catalyzes hydrolysis reaction, i.e. the addition of the hydrogen and hydroxyl ions of water to a molecule with its consequent splitting into two or more simpler molecules.
Proteolytic enzyme with a serine residue (Ser) in its active site. The reactivity of the serine residue is ensured by the vicinity of a histidine and an aspartate residue (catalytic triad), all three residues are required for the charge relay system to take place.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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