Recombinant human proteinase 3, the Wegener's autoantigen, expressed in HMC-1 cells is enzymatically active and recognized by c-ANCA

Ulrich Specks; David N. Fass; Michael P. Fautsch; Amber M. Hummel; Margaret A. Viss

doi:10.1016/0014-5793(96)00669-2

Recombinant human proteinase 3, the Wegener's autoantigen, expressed in HMC-1 cells is enzymatically active and recognized by c-ANCA

Ulrich Specks, David N. Fass, Michael P. Fautsch, Amber M. Hummel, Margaret A. Viss

Research output: Contribution to journal › Article › peer-review

41 Scopus citations

Abstract

We developed a stable expression system for conformationally intact recombinant human PR3 (rPR3) using the human mast cell line HMC-1. Like in U937 cells, the rPR3 is processed from a 34 kDa precursor to the 29 kDa mature form, primarily as the result of oligosaccharide trimming. The rPR3 binds [³H]DFP and hydrolyzes the substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-pNA. The enzymatic activity is inhibited by greater than 95% by α1-PI. The rPR3 and the enzymatically inactive mutant rPR3-S176A are both packaged in granules. Thus, proteolytic autoprocessing is not required for PR3's targeting to granules. This rPR3 is the first to be recognized by most c-ANCA from WG patients and all anti-PR3 ANCA that were detected by standard anti-PR3 specific ELISA. This expression system for rPR3 represents a versatile tool for the analysis of its intracellular processing, structure-function relationships and interaction with autoantibodies.

Original language	English (US)
Pages (from-to)	265-270
Number of pages	6
Journal	FEBS Letters
Volume	390
Issue number	3
DOIs	https://doi.org/10.1016/0014-5793(96)00669-2
State	Published - Jul 29 1996

Keywords

Anti-neutrophil cytoplasmic antibody
Expression system
Human
Mast cell
Neutral serine protease
Proteinase 3

ASJC Scopus subject areas

Biophysics
Structural Biology
Biochemistry
Molecular Biology
Genetics
Cell Biology

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@article{f3284b3f7b2a4bb4899fa295971b09b3,

title = "Recombinant human proteinase 3, the Wegener's autoantigen, expressed in HMC-1 cells is enzymatically active and recognized by c-ANCA",

abstract = "We developed a stable expression system for conformationally intact recombinant human PR3 (rPR3) using the human mast cell line HMC-1. Like in U937 cells, the rPR3 is processed from a 34 kDa precursor to the 29 kDa mature form, primarily as the result of oligosaccharide trimming. The rPR3 binds [3H]DFP and hydrolyzes the substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-pNA. The enzymatic activity is inhibited by greater than 95% by α1-PI. The rPR3 and the enzymatically inactive mutant rPR3-S176A are both packaged in granules. Thus, proteolytic autoprocessing is not required for PR3's targeting to granules. This rPR3 is the first to be recognized by most c-ANCA from WG patients and all anti-PR3 ANCA that were detected by standard anti-PR3 specific ELISA. This expression system for rPR3 represents a versatile tool for the analysis of its intracellular processing, structure-function relationships and interaction with autoantibodies.",

keywords = "Anti-neutrophil cytoplasmic antibody, Expression system, Human, Mast cell, Neutral serine protease, Proteinase 3",

author = "Ulrich Specks and Fass, {David N.} and Fautsch, {Michael P.} and Hummel, {Amber M.} and Viss, {Margaret A.}",

note = "Funding Information: AcknowledgementWs.{"}e thank Mr. JamesT araraf or help with the laser confocalm icroscopy,D r. Henry A. Homburgefro r the c-ANCA positivea ndc ontrolp atienst era,a ndMs. Kathy L. Stanke for experth elpwith the preparatioonf the manuscripWt. e are in-debtedt o Dr. E. Csernok,B ad BramstedtG, ermanya, nd Dr. J. Wieslander,L und, Sweden,f or the kind gift of antibodiesT. his work wasp erformeddu ringt het enureo f a Clinician-ScientAiswt ard from the AmericanH eartA ssociationto U.S., and supportebd y a ResearchG rantf romthe AmericanL ungA ssociationo f Minnesota (to U.S.) as well as fundsf romthe Mayo Foundation.",

year = "1996",

month = jul,

day = "29",

doi = "10.1016/0014-5793(96)00669-2",

language = "English (US)",

volume = "390",

pages = "265--270",

journal = "FEBS Letters",

issn = "0014-5793",

publisher = "Elsevier",

number = "3",

}

TY - JOUR

T1 - Recombinant human proteinase 3, the Wegener's autoantigen, expressed in HMC-1 cells is enzymatically active and recognized by c-ANCA

AU - Specks, Ulrich

AU - Fass, David N.

AU - Fautsch, Michael P.

AU - Hummel, Amber M.

AU - Viss, Margaret A.

N1 - Funding Information: AcknowledgementWs."e thank Mr. JamesT araraf or help with the laser confocalm icroscopy,D r. Henry A. Homburgefro r the c-ANCA positivea ndc ontrolp atienst era,a ndMs. Kathy L. Stanke for experth elpwith the preparatioonf the manuscripWt. e are in-debtedt o Dr. E. Csernok,B ad BramstedtG, ermanya, nd Dr. J. Wieslander,L und, Sweden,f or the kind gift of antibodiesT. his work wasp erformeddu ringt het enureo f a Clinician-ScientAiswt ard from the AmericanH eartA ssociationto U.S., and supportebd y a ResearchG rantf romthe AmericanL ungA ssociationo f Minnesota (to U.S.) as well as fundsf romthe Mayo Foundation.

PY - 1996/7/29

Y1 - 1996/7/29

N2 - We developed a stable expression system for conformationally intact recombinant human PR3 (rPR3) using the human mast cell line HMC-1. Like in U937 cells, the rPR3 is processed from a 34 kDa precursor to the 29 kDa mature form, primarily as the result of oligosaccharide trimming. The rPR3 binds [3H]DFP and hydrolyzes the substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-pNA. The enzymatic activity is inhibited by greater than 95% by α1-PI. The rPR3 and the enzymatically inactive mutant rPR3-S176A are both packaged in granules. Thus, proteolytic autoprocessing is not required for PR3's targeting to granules. This rPR3 is the first to be recognized by most c-ANCA from WG patients and all anti-PR3 ANCA that were detected by standard anti-PR3 specific ELISA. This expression system for rPR3 represents a versatile tool for the analysis of its intracellular processing, structure-function relationships and interaction with autoantibodies.

AB - We developed a stable expression system for conformationally intact recombinant human PR3 (rPR3) using the human mast cell line HMC-1. Like in U937 cells, the rPR3 is processed from a 34 kDa precursor to the 29 kDa mature form, primarily as the result of oligosaccharide trimming. The rPR3 binds [3H]DFP and hydrolyzes the substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-pNA. The enzymatic activity is inhibited by greater than 95% by α1-PI. The rPR3 and the enzymatically inactive mutant rPR3-S176A are both packaged in granules. Thus, proteolytic autoprocessing is not required for PR3's targeting to granules. This rPR3 is the first to be recognized by most c-ANCA from WG patients and all anti-PR3 ANCA that were detected by standard anti-PR3 specific ELISA. This expression system for rPR3 represents a versatile tool for the analysis of its intracellular processing, structure-function relationships and interaction with autoantibodies.

KW - Anti-neutrophil cytoplasmic antibody

KW - Expression system

KW - Human

KW - Mast cell

KW - Neutral serine protease

KW - Proteinase 3

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U2 - 10.1016/0014-5793(96)00669-2

DO - 10.1016/0014-5793(96)00669-2

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C2 - 8706874

AN - SCOPUS:0030605891

VL - 390

SP - 265

EP - 270

JO - FEBS Letters

JF - FEBS Letters

SN - 0014-5793

IS - 3

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