Structural and functional analysis of glycosylated Cu/Zn-superoxide dismutase from the fungal strain Humicola lutea 103.
The fungal strain Humicola lutea 103 produces a naturally glycosylated Cu/Zn-superoxide dismutase (Cu/ZnSOD) (HLSOD). To improve its yield, the effect of increased concentration of Cu2+ (from 1 to 750 microg/ml) on growth and enzyme biosynthesis was studied. The primary structure of this fungal enzyme has been determined by Edman degradation of peptide fragments derived from proteolytic digest. A single chain of the protein, consisting of 152 amino acid residues, reveals a very high degree (74-85%) of structural homology in comparison to the amino acid sequences of other fungal Cu/ZnSODs. The difference of the molecular masses of H. lutea Cu/ZnSOD, measured by MALDI-MS (15,935 Da) and calculated by its amino acid sequence (15,716 Da), is attributed to the carbohydrate chain of one mole of N-acetylglucosamine, attached to the N-glycosylation site Asn23-Glu-Ser. HLSOD protected mice from mortality after experimental influenza A/Aichi/2/68 (H3N2) virus infection. Using the glycosylated HLSOD, the survival rate is increased by 66% (protective index=86.1%) and the survival time prolonged by 5.2 days, similar to the application of ribavarin, while non-glycosylated bovine SOD conferred lower protection.
Dolashka-Angelova
Pavlina
P
Institute of Organic Chemistry, Bulgarian Academy of Sciences, Academician G. Bonchev 9, 1113 Sofia, Bulgaria. pda54@yahoo.com
Stevanovic
Stefan
S
Dolashki
Alexandar
A
Angelova
Maria
M
Serkedjieva
Julia
J
Krumova
Ekaterina
E
Pashova
Svetla
S
Zacharieva
Siika
S
Voelter
Wolfgang
W
D000595:Amino Acid Sequence; D000818:Animals; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D018533:Biomass; D002417:Cattle; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry / Q000633:toxicity; D004195:Disease Models, Animal; D005260:Female; D006031:Glycosylation; D009980:Influenza A virus / Q000187:drug effects; D007527:Isoenzymes; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D009976:Orthomyxoviridae Infections / Q000188:drug therapy; D021122:Protein Subunits; D012254:Ribavirin / Q000494:pharmacology; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013329:Structure-Activity Relationship; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism
D007527:Isoenzymes; D021122:Protein Subunits; D012254:Ribavirin; D003300:Copper; D013482:Superoxide Dismutase; D014357:Trypsin