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The precursor to the germ cell-specific PCSK4 proteinase is inefficiently activated in transfected somatic cells: evidence of interaction with the BiP chaperone

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Abstract

Proprotein convertase subtilisin/kexin type 4 (PCSK4), also known as proprotein convertase 4 (PC4), is a serine endoproteinase primarily expressed in testicular germ cells and in sperm. Inactivation of its gene in mouse causes male infertility. From studies of the biosynthesis of PCSK3/furin, its closest relative, it has been inferred that PCSK4 is synthesised in the endoplasmic reticulum as a zymogen; that it is rapidly matured by autocatalytic cleavage between the prodomain and the catalytic domain; that the cleaved prodomain remains attached to the mature enzyme; and that the enzyme is finally activated by the removal of the prodomain peptides following a secondary cleavage within the prodomain. In this study, we used human embryonic kidney 293 (HEK293) cells to study the biosynthesis of rat or human PCSK4. Our results show that the bulk of PCSK4 remains as an intracellular zymogen, presumably trapped in the endoplasmic reticulum, where it interacts with the general molecular chaperone glucose-regulated protein 78/Immunoglobulin heavy-chain binding protein (GRP78/BiP). These data suggest that, unlike other members of the convertase family, proPCSK4 cannot efficiently self-activate in somatic cells. These cells may lack the intracellular environment and the interacting molecules specific to testicular germ cells where this enzyme is normally expressed.

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Abbreviations

Aa:

Amino acid

BiP:

Immunoglobulin heavy-chain binding protein

CT:

Carboxyl-terminal

DMEM:

Dulbecco’s Modified Eagle’s Medium

ER:

Endoplasmic reticulum

FA:

Formic acid

FBS:

Foetal bovine serum

GlcNAc:

N-acetyl glucosamine

GRP:

Glucose-regulated protein

h-, m-, r-PCSK4:

Human, mouse, rat PCSK4

HEK:

Human embryonic kidney

HPLC:

High-performance liquid chromatography

IB:

Immunoblotting

IP:

Immunoprecipitation

IP/IB:

IP followed by IB

LC–MS/MS:

Liquid chromatograph tandem mass spectrometry

MCS:

Multiple cloning site

NARC-1:

Neural apoptosis regulated convertase-1

ORF:

Open reading frame

PBS:

Phosphate buffered saline

PC:

Proprotein convertase

PCSK:

Proprotein convertase subtilisin/kexin type

PNGase F:

Peptide-N4-(acetyl-β-glucosaminyl) aspargine amidase

PVDF:

Polyvinylidene fluoride

SDS-PAGE:

Sodium dodecyl sulphate polyacrylamide gel electrophoresis

SKI-1/S1P:

Subtilisin-kexin isozyme-1/site 1 protease

TBS:

Tris-buffered saline

TM:

Transmembrane

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Acknowledgments

The authors thank Mrs. Adriana Gambarotta, Mr. Pierre Bradley, and Mr. Garnet Rodger for their help in mouse maintenance and rabbit vaccination; Mrs. Haidy Tadros for the production of the two initial rPCSK4 expression vectors. We are grateful to Dr. Masahito Ikawa of Osaka University in Japan for his kind gift of testicular and sperm extracts from wild-type and calmegin-null mice. This work was funding by a grant from the Natural Sciences and Engineering Research Council of Canada.

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Authors and Affiliations

  1. Chronic Disease Program, Ottawa Hospital Research Institute, 725 Parkdale Avenue, Ottawa, ON, K1Y 4E9, Canada

    Charles Gyamera-Acheampong, Francine Sirois, Priyambada Mishra, Ajoy Basak & Majambu Mbikay

  2. Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada

    Charles Gyamera-Acheampong, Nicholas J. Denis, Priyambada Mishra, Daniel Figeys, Ajoy Basak & Majambu Mbikay

  3. Institute of Systems Biology, University of Ottawa, Ottawa, ON, Canada

    Nicholas J. Denis & Daniel Figeys

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  1. Charles Gyamera-Acheampong
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  2. Francine Sirois
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  7. Majambu Mbikay
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Correspondence to Majambu Mbikay.

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Gyamera-Acheampong, C., Sirois, F., Denis, N.J. et al. The precursor to the germ cell-specific PCSK4 proteinase is inefficiently activated in transfected somatic cells: evidence of interaction with the BiP chaperone. Mol Cell Biochem 348, 43–52 (2011). https://doi.org/10.1007/s11010-010-0635-y

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