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Two novel routes of transporter associated with antigen processing (TAP)-independent major histocompatibility complex class I antigen processing.

Snyder HL, Bacík I, Bennink JR, Kearns G, Behrens TW, Bächi T, Orlowski M, Yewdell JW - J. Exp. Med. (1997)

Bottom Line: We found that Jaw1 can efficiently deliver a COOH-terminal antigenic peptide to class I molecules in transporter associated with antigen processing (TAP)-deficient cells or cells in which TAP is inactivated by the ICP47 protein.Use of different protease inhibitors revealed the involvement of a nonproteasomal protease in the TAP-independent activity of cytosolic Jaw1.These findings demonstrate two novel TAP-independent routes of antigen processing; one based on highly efficient peptide liberation from the COOH terminus of membrane proteins in the ER, the other on delivery of a cytosolic protein to the ER by an unknown route.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892-0440, USA.

ABSTRACT
Jaw1 is an endoplasmic reticulum (ER) resident protein representative of a class of proteins post translationally inserted into membranes via a type II membrane anchor (cytosolic NH2 domain, lumenal COOH domain) in a translocon-independent manner. We found that Jaw1 can efficiently deliver a COOH-terminal antigenic peptide to class I molecules in transporter associated with antigen processing (TAP)-deficient cells or cells in which TAP is inactivated by the ICP47 protein. Peptide delivery mediated by Jaw1 to class I molecules was equal or better than that mediated by the adenovirus E3/19K glycoprotein signal sequence, and was sufficient to enable cytofluorographic detection of newly recruited thermostabile class I molecules at the surface of TAP-deficient cells. Deletion of the transmembrane region retargeted Jaw1 from the ER to the cytosol, and severely, although incompletely, abrogated its TAP-independent peptide carrier activity. Use of different protease inhibitors revealed the involvement of a nonproteasomal protease in the TAP-independent activity of cytosolic Jaw1. These findings demonstrate two novel TAP-independent routes of antigen processing; one based on highly efficient peptide liberation from the COOH terminus of membrane proteins in the ER, the other on delivery of a cytosolic protein to the ER by an unknown route.

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Rescue of Kd cell surface expression. T2-Kd cells were infected with the rVV indicated and the amount of cell surface Kd present  on viable cells cytofluorographically determined using a directly conjugated Kd-specific mAb. Data are expressed as mean channel fluorescence  × 100. The last three bars on the right represent cells coinfected with  Jaw1[NP147–155] and the rVV indicated (ACE, angiotensin-converting enzyme).
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Figure 4: Rescue of Kd cell surface expression. T2-Kd cells were infected with the rVV indicated and the amount of cell surface Kd present on viable cells cytofluorographically determined using a directly conjugated Kd-specific mAb. Data are expressed as mean channel fluorescence × 100. The last three bars on the right represent cells coinfected with Jaw1[NP147–155] and the rVV indicated (ACE, angiotensin-converting enzyme).

Mentions: In T2 cells, both Jaw1(Lum−)[NP147–155] (Fig. 3) and Jaw1[NP147–155] (data not shown) were efficiently presented after short VV infections (<4 h). This cannot be attributed to leakiness of the cells for cytosolic peptides, because under the same conditions, the cytosolic minigene, that is produced in enormous amounts relative to the amount of peptide liberated in the cytosol from proteins (36), was not presented above background values obtained with a control VV. [NP147–155] Jaw1-infected cells were recognized by NP-specific TCD8+ at levels observed using cells infected with control rVVs. This is consistent with the predicted topology of Jaw1, but we cannot eliminate the possibility that the secretory pathway, like the cytosol, is incapable of liberating the NP147–155 peptide from the NH2 terminus of Jaw1. NP147–155 was liberated from Jaw1(TM−)[NP147–155], but at far lower efficiency than from the transmembrane domain containing molecules (see Figs. 4 and 5).


Two novel routes of transporter associated with antigen processing (TAP)-independent major histocompatibility complex class I antigen processing.

Snyder HL, Bacík I, Bennink JR, Kearns G, Behrens TW, Bächi T, Orlowski M, Yewdell JW - J. Exp. Med. (1997)

Rescue of Kd cell surface expression. T2-Kd cells were infected with the rVV indicated and the amount of cell surface Kd present  on viable cells cytofluorographically determined using a directly conjugated Kd-specific mAb. Data are expressed as mean channel fluorescence  × 100. The last three bars on the right represent cells coinfected with  Jaw1[NP147–155] and the rVV indicated (ACE, angiotensin-converting enzyme).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199067&req=5

Figure 4: Rescue of Kd cell surface expression. T2-Kd cells were infected with the rVV indicated and the amount of cell surface Kd present on viable cells cytofluorographically determined using a directly conjugated Kd-specific mAb. Data are expressed as mean channel fluorescence × 100. The last three bars on the right represent cells coinfected with Jaw1[NP147–155] and the rVV indicated (ACE, angiotensin-converting enzyme).
Mentions: In T2 cells, both Jaw1(Lum−)[NP147–155] (Fig. 3) and Jaw1[NP147–155] (data not shown) were efficiently presented after short VV infections (<4 h). This cannot be attributed to leakiness of the cells for cytosolic peptides, because under the same conditions, the cytosolic minigene, that is produced in enormous amounts relative to the amount of peptide liberated in the cytosol from proteins (36), was not presented above background values obtained with a control VV. [NP147–155] Jaw1-infected cells were recognized by NP-specific TCD8+ at levels observed using cells infected with control rVVs. This is consistent with the predicted topology of Jaw1, but we cannot eliminate the possibility that the secretory pathway, like the cytosol, is incapable of liberating the NP147–155 peptide from the NH2 terminus of Jaw1. NP147–155 was liberated from Jaw1(TM−)[NP147–155], but at far lower efficiency than from the transmembrane domain containing molecules (see Figs. 4 and 5).

Bottom Line: We found that Jaw1 can efficiently deliver a COOH-terminal antigenic peptide to class I molecules in transporter associated with antigen processing (TAP)-deficient cells or cells in which TAP is inactivated by the ICP47 protein.Use of different protease inhibitors revealed the involvement of a nonproteasomal protease in the TAP-independent activity of cytosolic Jaw1.These findings demonstrate two novel TAP-independent routes of antigen processing; one based on highly efficient peptide liberation from the COOH terminus of membrane proteins in the ER, the other on delivery of a cytosolic protein to the ER by an unknown route.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892-0440, USA.

ABSTRACT
Jaw1 is an endoplasmic reticulum (ER) resident protein representative of a class of proteins post translationally inserted into membranes via a type II membrane anchor (cytosolic NH2 domain, lumenal COOH domain) in a translocon-independent manner. We found that Jaw1 can efficiently deliver a COOH-terminal antigenic peptide to class I molecules in transporter associated with antigen processing (TAP)-deficient cells or cells in which TAP is inactivated by the ICP47 protein. Peptide delivery mediated by Jaw1 to class I molecules was equal or better than that mediated by the adenovirus E3/19K glycoprotein signal sequence, and was sufficient to enable cytofluorographic detection of newly recruited thermostabile class I molecules at the surface of TAP-deficient cells. Deletion of the transmembrane region retargeted Jaw1 from the ER to the cytosol, and severely, although incompletely, abrogated its TAP-independent peptide carrier activity. Use of different protease inhibitors revealed the involvement of a nonproteasomal protease in the TAP-independent activity of cytosolic Jaw1. These findings demonstrate two novel TAP-independent routes of antigen processing; one based on highly efficient peptide liberation from the COOH terminus of membrane proteins in the ER, the other on delivery of a cytosolic protein to the ER by an unknown route.

Show MeSH
Related in: MedlinePlus