HtrA1 Proteolysis of ApoE In Vitro Is Allele Selective

Abstract

Apolipoprotein E (ApoE) belongs to a large class of proteins that solubilize lipids for physiological transport. Humans have three different APOE alleles—APOE ε2, APOE ε3 and APOE ε4—and genetic studies identified ApoE4 as the strongest genetic risk factor for Alzheimer’s disease (AD). People who are homozygous for ApoE4 (i.e. ApoE4/E4) are an order of magnitude more likely to develop late-onset AD (LOAD) than ApoE3/E3 carriers. Several differences between ApoE3 and ApoE4 may contribute to AD including the observation that ApoE4 is degraded to a greater extent than ApoE3 in the human brain. Experiments with high-temperature requirement serine peptidase A1 (HtrA1), which is found in the nervous system, demonstrate that HtrA1 is an allele-selective ApoE-degrading enzyme that degrades ApoE4 more quickly than ApoE3. This activity is specific to HtrA1, as similar assays with HtrA2 showed minimal ApoE4 proteolysis and trypsin had no preference between ApoE4 and ApoE3. HtrA1 has also been reported to cleave the tau protein (Tau) and the amyloid protein precursor (APP) to hinder the formation of toxic amyloid deposits associated with AD. Competition assays with ApoE4, ApoE3, and Tau revealed that ApoE4 inhibits Tau degradation. Thus, the identification of ApoE4 as an in vitro HtrA1 substrate suggests a potential biochemical mechanism that links ApoE4 regulation of AD proteins such as Tau.

The serine protease HtrA1 contributes to the formation of an extracellular 25-kDa apolipoprotein E fragment that stimulates neuritogenesis

“…28 kDa) were not generated (22). Furthermore, the apoE 25 kDa fragment was more rapidly degraded further by HtrA1 to smaller fragments when the apoE4 recombinant protein was assessed, as compared with apoE3 recombinant protein (22). This result is strikingly similar to what has been observed in human post-mortem brain tissues, in which carriers of the APOEe3/3 genotype had increased levels of the apoE 25 kDa fragment as compared with APOEe4/4 carriers (14).…”

“…Intriguingly, in these studies, larger fragments of apoE (e.g. 28 kDa) were not generated (22). Furthermore, the apoE 25 kDa fragment was more rapidly degraded further by HtrA1 to smaller fragments when the apoE4 recombinant protein was assessed, as compared with apoE3 recombinant protein (22).…”

“…Consistent with the latter idea, rat hippocampal neurons secrete a serine protease that generates apoE proteolytic fragments with a very similar MW profile to the fragments seen in human postmortem brain samples (9). Although the identity of the secreted enzyme responsible for the production of apoE proteolytic fragments by rat hippocampal neurons was not identified (9), a serine protease, high-temperature requirement serine peptidase A1 (HtrA1), has recently been shown to cleave recombinant apoE into fragments, including a predominant N-terminal apoE 25 kDa fragment (22), which again is remarkably similar to the apoE fragmentation pattern seen in human postmortem brain samples (9,14).…”

“…In a recent study using recombinant proteins, the serine protease HtrA1 was shown to produce apoE N-terminal fragments in an apoE isoform-dependent manner (22). Indeed, the HtrA1-mediated proteolysis of recombinant apoE3 produced a major apoE N-terminal fragment of ~25 kDa that was shown to encompass amino acid residues 1-195 (22).…”

“…6), we next assessed the role of HtrA1 in apoE 25 kDa fragment formation using HtrA1 boronic acid inhibitor (HBAI, Fig. 6A), as a highly specific HtrA1 inhibitor (22). Based on previous studies (22), we used HBAI at a concentration of 1 µM.…”