Abstract
The human serine protease high temperature requirement A1 (HTRA1) is highly expressed in the placental tissue, especially in the last trimester of gestation. This suggests that HTRA1 is involved in placental formation and function. With the aim of a better understanding of the role of HTRA1 in the placenta, candidate substrates were screened in a placenta protein extract using a gel-based mass spectrometric approach. Protease inhibitor alpha-1-antitrypsin, actin cytoplasmic 1, tropomyosin beta chain and ten further proteins were identified as candidate substrates of HTRA1. Among the identified candidate substrates, alpha-1-antitrypsin (A1AT) was considered to be of particular interest because of its important role as protease inhibitor. For investigation of alpha-1-antitrypsin as substrate of HTRA1 synthetic peptides covering parts of the sequence of alpha-1-antitrypsin were incubated with HTRA1. By mass spectrometry a specific cleavage site was identified after met-382 (AIPM382↓383SIPP) within the reactive centre loop of alpha-1-antitrypsin, resulting in a C-terminal peptide comprising 36 amino acids. Proteolytic removal of this peptide from alpha-1-antitrypsin results in a loss of its inhibitor function. Beside placental alpha-1-antitrypsin the circulating form in human plasma was also significantly degraded by HTRA1. Taken together, our data suggest a link between the candidate substrates alpha-1-antitrypsin and the function of HTRA1 in the placenta in the syncytiotrophoblast, the cell layer attending to maternal blood in the villous tree of the human placenta. Data deposition: Mass spectrometry (MS) data have been deposited to the ProteomeXchange with identifier PXD000473.
Highlights
Human high temperature requirement A1 (HTRA1) belongs to the HtrA serine protease family
The concentration of enriched HTRA1 was estimated by Western blot using a monoclonal HTRA1 antibody and applying known amount of recombinant HTRA1 expressed in insect cells (Fig. 1)
150 ng full-HTRA1 was extracted from 15 mg placenta proteins
Summary
Human HTRA1 belongs to the HtrA (high temperature requirement A) serine protease family. HtrA was first identified in Escherichia coli (E. coli) and is essential for the bacterial survival at high temperature [1]. The bacterial protease acts as a chaperone and degrades misfolded proteins at elevated temperatures [2]. The human protease HTRA1 was first isolated from SV40-transformed fibroblast where it has been identified as a down-regulated gene [3]. Crystal structure of HTRA1 shows that HTRA1 crystallises as a trimer [5]. This oligomerization may play a role in proteolytic activity of the protease, as observed by E. coli HTRA [6]
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