A novel L-ficolin/mannose-binding lectin chimeric molecule with enhanced activity against Ebola virus.
Ebola viruses constitute a newly emerging public threat because they cause rapidly fatal hemorrhagic fevers for which no treatment exists, and they can be manipulated as bioweapons. We targeted conserved N-glycosylated carbohydrate ligands on viral envelope surfaces using novel immune therapies. Mannose-binding lectin (MBL) and L-ficolin (L-FCN) were selected because they function as opsonins and activate complement. Given that MBL has a complex quaternary structure unsuitable for large scale cost-effective production, we sought to develop a less complex chimeric fusion protein with similar ligand recognition and enhanced effector functions. We tested recombinant human MBL and three L-FCN/MBL variants that contained the MBL carbohydrate recognition domain and varying lengths of the L-FCN collagenous domain. Non-reduced chimeric proteins formed predominantly nona- and dodecameric oligomers, whereas recombinant human MBL formed octadecameric and larger oligomers. Surface plasmon resonance revealed that L-FCN/MBL76 had the highest binding affinities for N-acetylglucosamine-bovine serum albumin and mannan. The same chimeric protein displayed superior complement C4 cleavage and binding to calreticulin (cC1qR), a putative receptor for MBL. L-FCN/MBL76 reduced infection by wild type Ebola virus Zaire significantly greater than the other molecules. Tapping mode atomic force microscopy revealed that L-FCN/MBL76 was significantly less tall than the other molecules despite similar polypeptide lengths. We propose that alterations in the quaternary structure of L-FCN/MBL76 resulted in greater flexibility in the collagenous or neck region. Similarly, a more pliable molecule might enhance cooperativity between the carbohydrate recognition domains and their cognate ligands, complement activation, and calreticulin binding dynamics. L-FCN/MBL chimeric proteins should be considered as potential novel therapeutics.
Michelow
Ian C
IC
From the Program of Developmental Immunology, Department of Pediatrics, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114.
Dong
Mingdong
M
Mungall
Bruce A
BA
Yantosca
L Michael
LM
Lear
Calli
C
Ji
Xin
X
Karpel
Marshall
M
Rootes
Christina L
CL
Brudner
Matthew
M
Houen
Gunnar
G
Eisen
Damon P
DP
Kinane
T Bernard
TB
Takahashi
Kazue
K
Stahl
Gregory L
GL
Olinger
Gene G
GG
Spear
Gregory T
GT
Ezekowitz
R Alan B
RA
Schmidt
Emmett V
EV
D000998:Antiviral Agents / Q000494:pharmacology*; D037282:Calreticulin / Q000737:chemistry; D045744:Cell Line, Tumor; D002626:Chemistry, Pharmaceutical / Q000379:methods; D003165:Complement System Proteins / Q000737:chemistry; D015195:Drug Design; D029043:Ebolavirus / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D037102:Lectins / Q000737:chemistry*; D037601:Mannose-Binding Lectin / Q000737:chemistry*; D018625:Microscopy, Atomic Force / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods; D000097604:Ficolins
D000998:Antiviral Agents; D037282:Calreticulin; D003165:Complement System Proteins; D037102:Lectins; D037601:Mannose-Binding Lectin; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000097604:Ficolins