Purification, cloning, and expression of human plasma hyaluronidase.
Hyaluronidase was purified from human plasma using Triton X-114 phase extractions and ion-exchange chromatography. Monoclonal antibodies generated against the purified protein by a novel screening assay were utilized to isolate homogeneous enzyme for microsequencing. The amino acid sequences obtained matched a cDNA in the Expressed Sequence Tag database which, with 5'-RACE-PCR, was used to clone the plasma hyaluronidase gene, termed Hyal-1. Hyal-1 codes for a protein of 435 amino acids that is over 40% identical to PH-20, a sperm-specific hyaluronidase. Unlike PH-20, which is only expressed in testis, transcripts of Hyal-1 were found in multiple tissues. Hyal-1 stably expressed in human embryonic kidney cells resulted in a 3,000 fold increase of secreted immunoreactive hyaluronidase activity that was biochemically indistinguishable from human plasma hyaluronidase. By immunological, molecular and biochemical criteria, we conclude that Hyal-1 is the predominant hyaluronidase found in human plasma.
Frost
G I
GI
Department of Pathology, School of Medicine, University of California, San Francisco 94143-0506, USA.
Csóka
A B
AB
Wong
T
T
Stern
R
R
Csóka
T B
TB
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D017421:Sequence Analysis
D018076:DNA, Complementary; D006821:Hyaluronoglucosaminidase