Molecular Studies Define the Primary Structure of α1-Antichymotrypsin (ACT) Protease Inhibitor in Alzheimer's Disease Brains: COMPARISON OF ACT IN HIPPOCAMPUS AND LIVER

Shin-Rong Hwang,Brent Steineckert,Andrea Kohn,Miklos Palkovits,Vivian Y.H Hook

doi:10.1074/jbc.274.3.1821

Abstract

An alpha1-antichymotrypsin-like serpin has been implicated in Alzheimer's disease (AD) based on immunochemical detection of alpha1-antichymotrypsin (ACT) in amyloid plaques from the hippocampus of AD brains. The presence of neuroendocrine isoforms of ACTs and reported variations in human liver ACT cDNA sequences raise the question of the molecular identity of ACT in brain. In this study, direct reverse transcription-polymerase chain reaction and cDNA sequencing indicate that the hippocampus ACT possesses the reactive site loop that is characteristic of serpins, with Leu as the predicted P1 residue interacting with putative chymotrypsin-like target proteases. The deduced primary sequence of the human hippocampus ACT possesses more than 90% homology with reported primary sequences for the human liver ACT. Moreover, identical ACT primary sequences deduced from the cDNAs were demonstrated in the hippocampus of control and AD brains. Northern blots showed that ACT mRNA expression in hippocampus was 900 times lower than that in liver. Also, hippocampus and liver ACT proteins demonstrated differential sensitivities to deglycosylation. Overall, reverse transcription-polymerase chain reaction combined with cDNA and primary sequence analyses have defined the molecular identity of human hippocampus ACT in control and AD brains. The determined reactive site loop domain of hippocampus ACT will allow prediction of potential target proteases inhibited by ACT in AD.

Highlights

From the ‡Department of Medicine and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0822, ¶Department of Biochemistry, Uniformed Services University, Bethesda, Maryland 20892, and ʈFirst Department of Anatomy, Semmelweis University, Budapest, Hungary
Direct reverse transcriptionpolymerase chain reaction and cDNA sequencing indicate that the hippocampus ACT possesses the reactive site loop that is characteristic of serpins, with Leu as the predicted P1 residue interacting with putative chymotrypsin-like target proteases
reverse transcriptase polymerase chain reaction (RT-PCR) of Hippocampus ACT cDNA Defines the open reading frame (ORF) Encoding the ACT Primary Sequence—RT-PCR was used to determine the primary sequence of ACT expressed in hippocampus from Alzheimer’s disease (AD) and normal brains

Summary

Introduction

Direct reverse transcriptionpolymerase chain reaction and cDNA sequencing indicate that the hippocampus ACT possesses the reactive site loop that is characteristic of serpins, with Leu as the predicted P1 residue interacting with putative chymotrypsin-like target proteases. Reverse transcription-polymerase chain reaction combined with cDNA and primary sequence analyses have defined the molecular identity of human hippocampus ACT in control and AD brains. Variations in the deduced primary sequences of human liver ACT cDNAs have been reported [5,6,7,8] Because these ACT isoforms are all recognized by anti-ACT sera, these observations raise the question of the molecular identity of ACT-like immunoreactivity in Alzheimer’s disease brains. This study has, defined the primary sequence and characteristics of ACT expressed in control and Alzheimer’s disease brains

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Conclusion