Deletion in HCDR3 rescues T15 antibody mutants from a secretion defect caused by mutations in HCDR2.
We recently described mutants of the murine anti-phosphocholine Ab T15, with changes in heavy chain complementarity determining region 2 (HCDR2) that caused loss of secretion. Surprisingly, the T15 HCDR2 mutations did not alter secretion when placed into the related anti-phosphocholine Ab D16, which differs from T15 only in HCDR3 and light (L) chain. Here, we exploit the differences between these two Abs to assess the basis of the secretion defect. The T15 L chain is not secreted in the absence of heavy (H) chain. In contrast, D16 L chain is secreted in the absence of H chain, as are most L chains. We co-expressed the T15 wild-type (wt) and mutant H chains with the D16 L chain, as well as with another secreted L chain, J558L. The mutant H chains were not secreted when expressed with either heterologous L chain. These results establish that the T15 L chain is not uniquely associated with the defect. The T15 and D16 Abs also differ in HCDR3 length in that D16 lacks four amino acid residues (Ser99, Ser100, Tyr100a, Trp100b) present in T15. We deleted these four residues from T15 wt and mutant H chains. Secretion of T15 wt was unaffected by the deletion, but shortening HCDR3 restored secretion in the HCDR2 mutants regardless of L chain association. Together these data demonstrate that both the HCDR2 and HCDR3 domains contain structural information that may affect the secretion competence of Abs.
Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland 97201, USA.
T M
TM
Martin
C
C
Kowalczyk
S
S
Stevens
G D
GD
Wiens
M P
MP
Stenzel-Poore
M B
MB
Rittenberg
D000595:Amino Acid Sequence; D000818:Animals; D017152:Antibodies, Antiphospholipid / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation / Q000276:immunology*; D010767:Phosphorylcholine / Q000276:immunology*; D017384:Sequence Deletion / Q000276:immunology; D013489:Suppression, Genetic / Q000235:genetics*
D017152:Antibodies, Antiphospholipid; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D010767:Phosphorylcholine