Modulation of in vivo IgG crystallization in the secretory pathway by heavy chain isotype class switching and N-linked glycosylation.
Crystalline bodies (CBs) can develop in the endoplasmic reticulum (ER) of antibody-producing cells. Although this phenotype is often reported in association with plasma cell dyscrasias and other hematological disorders, the details of CB biogenesis and CB's roles in pathophysiology remain poorly understood. Using an imaging-based screening method, we identified a secretion-competent human IgG2/λ clone that develops spindle-shaped intracellular crystals in transiently-transfected HEK293 cells upon Brefeldin A treatment. When stably overexpressed from CHO cells, the IgG2/λ clone spontaneously produced spindle-shaped CBs in the ER. Some CBs were released to the extracellular space while remaining enclosed by the membranes of secretory pathway origin. Structural modeling on the variable-region did not uncover prominent surface characteristics such as charge clusters. In contrast, alterations to the constant domain-encoded properties revealed their modulatory roles in CB-inducing propensities and CB morphology. For example, deletion of the entire Fc domain changed the morphology of CBs into thin filaments. Elimination of an N-linked glycan by a N297A mutation promoted Russell body biogenesis accompanied by marked reduction in IgG secretion. Isotype class switching from the original IgG2 to IgG1 and IgG4 changed the crystal morphology from spindle-shaped to long needle and acicular shaped, respectively. The IgG3 version, in contrast, suppressed the CB formation. Either the HC or LC alone or the Fc-domain alone did not trigger CB biogenesis. An IgG's in vivo crystal morphology and crystallization propensity can thus be modulated by the properties genetically and biochemically encoded in the HC constant region.
Department of Therapeutic Discovery, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119, USA. Electronic address: harukih@amgen.com.
Haruki
H
Hasegawa
Department of Therapeutic Discovery, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119, USA.
Carla
C
Forte
Department of Therapeutic Discovery, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119, USA.
Irene
I
Barber
Department of Therapeutic Discovery, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119, USA.
Shanon
S
Turnbaugh
Department of Therapeutic Discovery, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119, USA.
Janelle
J
Stoops
Department of Therapeutic Discovery, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119, USA.
Min
M
Shen
Department of Therapeutic Discovery, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119, USA.
Ai Ching
AC
Lim
D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D016466:CHO Cells; D003412:Cricetulus; D003460:Crystallization; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D015870:Gene Expression; D006031:Glycosylation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D017578:Immunoglobulin Class Switching; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D008857:Microscopy, Interference; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D055571:Secretory Pathway / Q000235:genetics
D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D020126:Brefeldin A
Crystalline body; Endoplasmic reticulum; Immunoglobulin; Protein aggregation; Protein crystallization; Russell body