Zyme, a Novel and Potentially Amyloidogenic Enzyme cDNA Isolated from Alzheimer's Disease Brain

Abstract

The deposition of the ␤ amyloid peptide in neuritic plaques and cerebral blood vessels is a hallmark of Alzheimer's disease (AD) pathology. The major component of the amyloid deposit is a 4.2-kDa polypeptide termed amyloid ␤-protein of 39 -43 residues, which is derived from processing of a larger amyloid precursor protein (APP). It is hypothesized that a chymotrypsin-like enzyme is involved in the processing of APP.We have discovered a new serine protease from the AD brain by polymerase chain reaction amplification of DNA sequences representing active site homologous regions of chymotrypsin-like enzymes. A cDNA clone was identified as one out of one million that encodes Zyme, a serine protease. Messenger RNA encoding Zyme can be detected in some mammalian species but not in mice, rats, or hamster. Zyme is expressed predominantly in brain, kidney, and salivary gland. Zyme mRNA cannot be detected in fetal brain but is seen in adult brain. The Zyme gene maps to chromosome 19q13.3, a region which shows genetic linkage with late onset familial Alzheimer's disease.When Zyme cDNA is co-expressed with the APP cDNA in 293 (human embryonic kidney) cells, amyloidogenic fragments are detected using C-terminal antibody to APP. These co-transfected cells release an abundance of truncated amyloid ␤-protein peptide and shows a reduction of residues 17-42 of A␤ (P3) peptide. Zyme is immunolocalized to perivascular cells in monkey cortex and the AD brain. In addition, Zyme is localized to microglial cells in our AD brain sample. The amyloidogenic potential and localization in brain may indicate a role for this protease in amyloid precursor processing and AD.The generation of the ␤ amyloid peptide is thought to be the result of processing of the amyloid precursor protein (APP) 1 by one or more proteases. After the deduced amino acid sequence of APP was revealed, a number of laboratories initiated studies to purify and characterize the N-terminal cleaving enzyme of amyloid ␤-protein (A␤), termed ␤-secretase (1). The cleavage of the Met 596 -Asp 597 bond of the full-length APP generates the N-terminal amino acid of A␤, which was first shown by Glenner and Wong (2) to be aspartic acid. ␤-Secretase is yet an unidentified protease.Several themes and strategies influenced the direction of investigation of ␤-secretase. The first strategy was to follow a traditional biochemical purification. Assays were utilized in which short peptide substrates were substituted for the large transmembrane precursor protein (1). Any enzyme capable of making a methionine (M)/aspartic acid (D) cleavage could be designated a potential ␤-secretase. The second theme, since the amino acid that surrounded the N terminus of A␤ was found to be a methionine, was some laboratories have rationalized that a search for an enzyme with chymotrypsin-like specificity (a specificity for cleavage of subtrates containing a neutral hydrophobic residue at the S1 subsite) was necessary (3-7).To facilitate the second approach, we have developed a method to identify chymotrypsin-l...

Immunofluorometric assay of human kallikrein 6 (zyme/protease M/neurosin) and preliminary clinical applications

“…Previously, we have demonstrated presence of other kallikreins, including PSA and hK2, in these biologic fluids (20 -27). We have further detected large amounts of hK6 protein in cerebrospinal fluid, which are consistent with the observation that hK6 is expressed at high levels in brain tissue (7). We have further demonstrated presence of hK6 in male and female sera and seminal plasmas and in a small percentage of amniotic fluids and breast tumor cytosols.…”

“…15,16. IMMUNOFLUOROMETRIC ASSAY OF HUMAN KALLIKREIN 6 The newly developed immunofluorometric assay for hK6 may find applications toward the possible diagnostic/prognostic value of this biomarker in various human diseases. More specifically, we are interested to examine if the levels of hK6 in cerebrospinal fluid are associated with Alzheimer's or other degenerative neurologic diseases, as preliminarily suggested by Little et al (7). We are further interested to examine if hK6 concentration in serum or other biologic fluids is altered in various malignancies, with mechanisms similar to those operating for PSA and hK2.…”

“…The KLK6 gene (encoding for human kallikrein 6 [hK6]) has been cloned independently by three groups of investigators and was previously given the names zyme (7), protease M (8) and neurosin (9). The gene encodes for a trypsin-like serine protease of 244 amino acids in length, of which sixteen amino acids constitute the signal peptide and five amino acids the activation peptide.…”

Immunofluorometric quantitation and histochemical localisation of kallikrein 6 protein in ovarian cancer tissue: a new independent unfavourable prognostic biomarker

“…Previously called zyme (Little et al, 1997), protease M (Anisowicz et al, 1996), and neurosin (Yamashiro et al, 1997), kallikrein 6 is now the accepted name (Diamandis et al, 2000b). hK6 is a trypsin-like serine protease of unknown physiologic function that is secreted as a 223 amino acid protein.…”

“…Various tissues express the hK6 gene and synthesize the protein (Diamandis et al, 2000c;Yousef et al, 1999). Expression is particularly prominent in the brain (Yamashiro et al, 1997), and this, along with the amyloidogenic potential of hK6 (Little et al, 1997) has raised the possibility of a role for hK6 in amyloid precursor processing and the development of Alzheimer's disease. Recently, Diamandis et al (2000d) have shown that hK6 is elevated in the cerebrospinal fluid and blood of individuals with histologically confirmed Alzheimer's, thereby suggesting hK6 may be of value in the diagnosis of this disorder.…”