Request ID: cfg_rRequest_600
Status
:
Approved
Project Description
:
Carbohydrate-binding specificities of extracellular fragments of 3 influenza H5 variants
CFG Member
:
Other
Requester First Name
:
Hud
Requester Last Name
:
Freeze
Head of Lab First Name
:
Hud
Head of Lab Last Name
:
Freeze
Assigned First Name
:
Hudson H.
Assigned Last Name
:
Freeze
Requester Email
:
hudson@burnham.org
Requester Interest
:
Dr. Freeze's lab studies human congenital disorders of glycosylation, a group of over a dozen inherited diseases with broad multisystemic symptoms. This laboratory also studies nontraditional carboxylated N-linked glycans that mediate inflammation and uses an anti-glycan monoclonal antibody to block their development.
Request Date [yyyy-mm-dd]
:
2006-06-05
Institution
:
Burnham Institute for Medical Research
Shipping Address
:
858-646-3142
Comments
:
Information not entered/not applicable.
CFG Core
:
H
Resource Type
:
Information not entered/not applicable.
Amount Requested
:
Information not entered/not applicable.
Date that your RNA/GBP samples will be sent to the core [yyyy-mm-dd]
:
2025-01-15
Experiment to be conducted
:
H5N1, an avian influenza, is a growing pandemic threat. Glycosylation of hemagglutinin (HA) is known to reduce antigenicity, protect HA from proteolytic attack and modulate the affinity of HA for its host cell receptor, sialic acid. Avian influenza viruses infect the intestinal tract where they bind Sia2,3Gal, and human influenza viruses infect the upper respiratory tract where they bind Sia2,6Gal. This linkage preference acts both as a species barrier and to restrict the breadth of cell types that can become infected. Thus, the ability to bind both types of sialic acid linkages may both allow the virus to cross species barriers and to become a systemic infection.
Asn158 (H3 numbering) is the only polymorphic glycosylation site in H5. Glycosylation of Asn158, which overlooks the sialic acid binding pocket of HA, results in decreased affinity of HA for sialic acid (Ohuchi et al, 1997; Matrosovich et al, 1999). Also, the loss of glycosylation at Asn158 in H1N1 results in a switch from Sia2,3Gal to Sia2,6Gal ligand preference (Marinina et al, 2003). Glycosylation of this residue correlates with the shortening of the neuraminidase (NA) stalk. This stalk shortening results in reduced activity of NA (Castrucci and Kawaoka, 1993; Els et al, 1985; Matrosovich et al, 1999). Similarly, growth of influenza in the presence of neuraminidase inhibitors selects for glycosylation of residues near to the sialic acid binding pocket (Mishen et al, 2005). In the context of a shortened NA stalk, glycosylation near the sialic acid binding pocket is critical for efficient viral replication in cell culture (Wagner et al, 2000). These two changes, glycosylation and stalk shortening, have historically occurred upon adaptation of Influenza to chickens from wild aquatic birds. However, more recently these changes are being seen in wild aquatic birds, which are typically asymptomatic carriers, but which are now dying from infection with H5N1 (Guan et al, 2004).
Interestingly, the 2006 H5N1 strain isolates have aspartic acid at position 158. Substitution of Asn158 with glutamatic acid in H1N1 has been linked to a switch to a phenotype that replicates more efficiently (Both et al., 1983). In 2006 H5N1, this switch to an acidic residue occurs in conjunction with shortening of the NA stalk, suggesting that it may also result in decreased affinity of HA for sialic acid.
Why are these changes occurring in strains of H5N1? What is the advantage for the virus? Why reduce the affinity for sialic acid? We speculate that glycosylation of Asn158 or introduction of an acidic residue at this position modulates the specificity of HA. We, therefore, propose to determine the glycan array profile for polymorphic variants of the sequence surrounding Asn158. Specifically, we will test the following sequence variants: N158/S159/T160; N158/S159/A160; and D158/N159/A160. All three sequence variants have been seen in isolates of H5N1.
We have expressed the extracellular portion of H5 in a baculovirus system using the methodology in Stevens et al (2006). The recombinant protein is soluble, trimeric, HIS-tagged and >90% uncleaved. We have made the above mutants, and propose to use the established methodology to assay the glycan array profile. Since this was already published (Stevens et al, 2006) using CFG resources, it should pose no obvious challenge or problems.
Both, GW, Cheng H-S, and Kilbourne ED Hemagglutinin of swine influenza virus: a single amino acid change pleiotropically affects viral antigenicity and replication Proc Natl Acad Sci 80: 6996 (1983)
Castrucci MR and Kawaoka Y Biologic importance of neuraminidase stalk length in influenza A virus J Virol 67(2) 759-764 (1993)
Els MC, Air GM, Murti KG, Webster RG, and Laver WG An 18 amino acid deletion in an influenza neuraminidase Virology 142:241-248 (1985)
Guan Y, Poon LLM, Cheung CY, Ellis TM, Lim W. Lipatov AS, Chan KH, Sturm-Ramirez KM, Cheung CL, Leung YHC, Yeun KY, Webster RG
Within Scope of Consortium
:
Y
If yes, indicate the person responsible for inputing data into core B
:
Hud Freeze
GBP being Addressed
:
Avian influenza H5N1 hemagglutinin (HA) carrying alterations at Asn158, a polymophic glycosylation site in H5
Specifc aims being addressed
:
Define the specificity and affinity for carbohydrate ligands.