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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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Related in: MedlinePlus

Presentation of NP147–155 by rVV-infected cells. rVV-infected cells expressing the Jaw chimeric proteins were tested for lysis by NP-specific  secondary TCD8+ at the indicated effector to target ratio. Cells were coinfected with a rVV expressing Kd to enable recognition.
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Figure 3: Presentation of NP147–155 by rVV-infected cells. rVV-infected cells expressing the Jaw chimeric proteins were tested for lysis by NP-specific secondary TCD8+ at the indicated effector to target ratio. Cells were coinfected with a rVV expressing Kd to enable recognition.

Mentions: The antigen processing of Jaw1 constructs was studied in T2 cells (a TAP-deficient human lymphoid cell line) or L929 cells (a TAP-expressing mouse cell line). To enable recognition by Kd-restricted, NP-specific TCD8+, cells were coinfected with a rVV expressing Kd in addition to the Jaw1-expressing rVV. As seen in Fig. 3, L929 cells presented NP147–155 from the COOH termini of both Jaw1(Lum−)[NP147–155] and Jaw1(TM−)[NP147–155]. Presentation from the NH2 terminus of Jaw1 occurred at much lower levels. Because a Met-initiated cytosolic minigene product that is identical to the 10 NH4-terminal residues of [NP147–155] Jaw1 is effectively presented by L929 cells (designated in Fig. 3 and [NP147–155]), this indicates that the Jaw1-derived COOH-terminal flanking sequences cannot be efficiently removed from NP147–155 by cytosolic proteases. This compromises the use of this construct as a control in T2 cells, but only partially, because the activities of cytosolic proteases and secretory proteases are expected to be independent.


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)

Presentation of NP147–155 by rVV-infected cells. rVV-infected cells expressing the Jaw chimeric proteins were tested for lysis by NP-specific  secondary TCD8+ at the indicated effector to target ratio. Cells were coinfected with a rVV expressing Kd to enable recognition.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Presentation of NP147–155 by rVV-infected cells. rVV-infected cells expressing the Jaw chimeric proteins were tested for lysis by NP-specific secondary TCD8+ at the indicated effector to target ratio. Cells were coinfected with a rVV expressing Kd to enable recognition.
Mentions: The antigen processing of Jaw1 constructs was studied in T2 cells (a TAP-deficient human lymphoid cell line) or L929 cells (a TAP-expressing mouse cell line). To enable recognition by Kd-restricted, NP-specific TCD8+, cells were coinfected with a rVV expressing Kd in addition to the Jaw1-expressing rVV. As seen in Fig. 3, L929 cells presented NP147–155 from the COOH termini of both Jaw1(Lum−)[NP147–155] and Jaw1(TM−)[NP147–155]. Presentation from the NH2 terminus of Jaw1 occurred at much lower levels. Because a Met-initiated cytosolic minigene product that is identical to the 10 NH4-terminal residues of [NP147–155] Jaw1 is effectively presented by L929 cells (designated in Fig. 3 and [NP147–155]), this indicates that the Jaw1-derived COOH-terminal flanking sequences cannot be efficiently removed from NP147–155 by cytosolic proteases. This compromises the use of this construct as a control in T2 cells, but only partially, because the activities of cytosolic proteases and secretory proteases are expected to be independent.

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