HTRA1 Mutations Identified in Symptomatic Carriers Have the Property of Interfering the Trimer-Dependent Activation Cascade

Abstract

Background: Mutations in the high-temperature requirement A serine peptidase 1 (HTRA1) cause cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). Most carriers for HTRA1 mutations are asymptomatic, but more than 10 mutations have been reported in symptomatic carriers. The molecular differences between the mutations identified in symptomatic carriers and mutations identified only in CARASIL patients are unclear. HTRA1 is a serine protease that forms homotrimers, with each HTRA1 subunit activating the adjacent HTRA1 via the sensor domain of loop 3 (L3) and the activation domain of loop D (LD). Previously, we analyzed four HTRA1 mutant proteins identified in symptomatic carriers and found that they were unable to form trimers or had mutations in the LD or L3 domain. The mutant HTRA1s with these properties are presumed to inhibit trimer-dependent activation cascade. Indeed, these mutant HTRA1s inhibited wild-type (WT) protease activity. In this study, we further analyzed 15 missense HTRA1s to clarify the molecular character of mutant HTRA1s identified in symptomatic carriers. Methods: We analyzed 12 missense HTRA1s identified in symptomatic carriers (hetero-HTRA1) and three missense HTRA1s found only in CARASIL (CARASIL-HTRA1). The protease activity of the purified recombinant mutant HTRA1s was measured using fluorescein isothiocyanate-labeled casein as substrate. Oligomeric structure was evaluated by size-exclusion chromatography. The protease activities of mixtures of WT with each mutant HTRA1 were also measured. Results: Five hetero-HTRA1s had normal protease activity and were excluded from further analysis. Four of the seven hetero-HTRA1s and one of the three CARASIL-HTRA1s were unable to form trimers. The other three hetero-HTRA1s had mutations in the LD domain. Together with our previous work, 10 of 11 hetero-HTRA1s and two of six CARASIL-HTRA1s were either defective in trimerization or had mutations in the LD or L3 domain ( P = 0.006). By contrast, eight of 11 hetero-HTRA1s and two of six CARASIL-HTRA1 inhibited WT protease activity ( P = 0.162). Conclusions: HTRA1 mutations identified in symptomatic carriers have the property of interfering the trimer-dependent activation cascade of HTRA1.

HTRA1-Related Cerebral Small Vessel Disease: A Review of the Literature

“…The youngest and oldest reported ages at onset of neurological symptoms/signs among symptomatic carriers were 29 (25) and 77 (30), respectively, and the youngest and oldest ages at onset (7,18,26).…”

“…It thus remains unclear why certain mutations cause CSVD in HTRA1 carriers. Previously, we reported that either a deficiency in trimerization or an amino-acid mutation located in the loop D (LD) or loop 3 (L3) domain was common in missense HTRA1 proteins identified in symptomatic carriers (18). We speculated that these mutations in the HTRA1 gene may inhibit wild-type (WT) protease activity (7).…”

Cerebral Small Vessel Disease Related to a Heterozygous Nonsense Mutation in <i>HTRA1</i>

“…However, some mutations identified in manifested carriers did not show dominant-negative effects (4,5,11). In these cases, haploinsufficiency, which often results in reduced penetrance and variable expressivity, may explain the occurrence of cerebral SVD.…”

“…Indeed, several studies have experimentally demonstrated that certain missense mutant HTRA1 proteins show a marked decrease in protease activities as well as reduced wild-type HTRA1 activity, presumably due to interference in the trimer-dependent activation cascade of the enzyme. Recently, two different mechanisms were proposed to underlie the dominant-negative effects of HTRA1 mutations (4,11): the inhibition of trimer formation, and the inhibition of the wild-type HTRA1 activity, observed in several missense mutations located in the sensor domain of loop 3 (L3) or the activation domain of loop D (LD) of HTRA1. These two domains play an essential role in the normal protease activity of HTRA1 (10,12).…”

“…These results have important implications for the pathogenesis of the p.R 302X mutation. p.R302X was shown to result in the complete loss of protease activity (11); however, its ability to form trimers or express a dominant-negative effect has not yet been determined. In addition, Lee et al showed that p.Q289X and p.A 182PfsX33 induced the complete loss of protease activity and dominant-negative effects, although the exact mechanisms were not elucidated (5).…”