Regulation of CD44 binding to hyaluronan by glycosylation of variably spliced exons.
The hyaluronan (HA)-binding function (lectin function) of the leukocyte homing receptor, CD44, is tightly regulated. Herein we address possible mechanisms that regulate CD44 isoform-specific HA binding. Binding studies with melanoma transfectants expressing CD44H, CD44E, or with soluble immunoglobulin fusions of CD44H and CD44E (CD44H-Rg, CD44E-Rg) showed that although both CD44 isoforms can bind HA, CD44H binds HA more efficiently than CD44E. Using CD44-Rg fusion proteins we show that the variably spliced exons in CD44E, V8-V10, specifically reduce the lectin function of CD44, while replacement of V8-V10 by an ICAM-1 immunoglobulin domain restores binding to a level comparable to that of CD44H. Conversely, CD44 bound HA very weakly when exons V8-V10 were replaced with a CD34 mucin domain, which is heavily modified by O-linked glycans. Production of CD44E-Rg or incubation of CD44E-expressing transfectants in the presence of an O-linked glycosylation inhibitor restored HA binding to CD44H-Rg and to cell surface CD44H levels, respectively. We conclude that differential splicing provides a regulatory mechanism for CD44 lectin function and that this effect is due in part to O-linked carbohydrate moieties which are added to the Ser/Thr rich regions encoded by the variably spliced CD44 exons. Alternative splicing resulting in changes in protein glycosylation provide a novel mechanism for the regulation of lectin activity.
Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121, USA.
K L
KL
Bennett
B
B
Modrell
B
B
Greenfield
A
A
Bartolazzi
I
I
Stamenkovic
R
R
Peach
D G
DG
Jackson
F
F
Spring
A
A
Aruffo
D017398:Alternative Splicing / Q000502:physiology; D001483:Base Sequence; D050260:Carbohydrate Metabolism; D005091:Exons / Q000502:physiology*; D006031:Glycosylation; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000378:metabolism*; D037102:Lectins / Q000378:metabolism; D008545:Melanoma; D008969:Molecular Sequence Data; D014407:Tumor Cells, Cultured / Q000378:metabolism / Q000648:ultrastructure
D018960:Hyaluronan Receptors; D037102:Lectins; D006820:Hyaluronic Acid