Multiple heparanases are expressed in polymorphonuclear cells.
BACKGROUND
Heparan sulfate proteoglycans are complex cell surface molecules containing polysaccharides called heparan sulfate. Lysosomes, platelet granules, and neutrophils (polymorphonuclear cells) contain heparanases that degrade heparan sulfate. There are at least two groups of heparanases: connective tissue-activating-peptide (CTAP-III) and mammalian heparanase (hpa). The purpose of this study was to quantify the expression of both CTAP-III and hpa in neutrophils and their heparanase activity.


METHODS
Neutrophils were isolated from whole blood, total RNA collected, and reverse transcriptase--polymerase chain reaction (RT-PCR) performed. Primers were designed for CTAP-III and hpa-1 sequences from GenBank. Neutrophil lysate underwent Western blot analysis (and quantification) with antibodies to the C-terminus of CTAP-III and the 50-kDa subunit of hpa1. Chromatography separated these components of lysate, which were then tested for heparanase activity.


RESULTS
Both CTAP-III (281 bp) and hpa-1 (485 bp) messenger RNA (mRNA) were expressed equally by neutrophils with use of quantitative RT-PCR. By Western blot analysis, a CTAP-III-like protein was detected at 80 kDa, and hpa-1 was detected as a 50-kDa protein, with expression not significantly different (P > 0.05). Heparanase activity was significantly different (P < 0.0001) for the 50-kDa hpa-1 protein (1.51 x 10(-6) micromol/min) and the 80-kDa CTAP-III-like protein (0.85 x 10(-6) micromol/min).


CONCLUSIONS
Human neutrophils express mRNA and protein for both a CTAP-III-like protein and hpa-1. Although expressed in similar quantity for mRNA and protein, Hpa-1 was more active as heparanase than the CTAP-III-like protein. With more than one class of heparanase in their granules, neutrophils may be able to modify different kinds of heparan sulfate chains.
Kosir
Mary Ann
MA
Surgical Service, VA Medical Center, Detroit, Michigan 48201, USA. mary.kosir@med.va.gov
Foley-Loudon
Patricia A
PA
Finkenauer
Raphaela
R
Tennenberg
Steven D
SD
D000595:Amino Acid Sequence; D000906:Antibodies; D001779:Blood Coagulation Factors / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D015870:Gene Expression / Q000276:immunology; D019763:Heparin Lyase / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology*; D010455:Peptides; D012333:RNA, Messenger / Q000032:analysis; D013347:Subcellular Fractions / Q000201:enzymology
D000906:Antibodies; D001779:Blood Coagulation Factors; D019763:Heparin Lyase; D006497:Heparan Sulfate; D010455:Peptides; D012333:RNA, Messenger; C009862:connective tissue-activating peptide; C018329:low affinity platelet factor 4