PARENT SESSION
SLIDE SESSION 16: FERTILIZATION
Chairs: William Breed, Nuch Tanphaichitr, Becky Sortini (Trainee)
Univ Ottawa-UCU Auditorium
1:30 PM-3:30 PM
434
ENZYMATIC PROPERTIES OF PIG SPERM SURFACE ARYLSULSULFATASE A.
Shoushtarian, Ali1, Carmona, Euridice1, Tantibhedhyangkul, Julierut1, Buhr, Mary2, Tanphaichitr, Nongnuj1,3, 1 2 3
ABSTRACT- Our laboratory has shown by molecular cloning and tryptic peptide sequencing that P68, a pig sperm plasma membrane protein involved in zona pellucida (ZP)/sulfogalactosylglycerolipid (SGG) binding, is arylsulfatase A (AS-A). Since the derived peptide sequence contains all amino acids of the AS-A active site pocket, we investigated whether sperm surface AS-A contained enzymatic properties as other AS-A. Sperm surface AS-A, purified from sperm plasma membrane extracts, by chromatofocusing followed by Sephacryl S-200 size-exclusion chromatography, showed desulfation activity on an artificial substrate, p-nitrocatechol sulfate (NCS) and natural substrates, SGG and sulfogalactosylceramide (SGC) in a detergent containing buffer, with its pH optimum in the range 4.5 to 5.0, and Km of 7.02, 0.091 and 0.121 mM, for these three substrates, respectively. All of these properties were similar to those of human liver AS-A, the prototype enzyme. Since, AS-A is present on the sperm surface, intact sperm were expected to have NCS desulfation activity. Our results showed that NCS could be desulfated at 0.15 nmoles/h/million of live pig sperm at pH 5.0. The absence of propidium iodide (PI) staining in these sperm indicated that the membrane was still intact, suggesting that the AS-A activity observed was from the surface entity and not from the acrosome, which also contains AS-A. Since AS-A does not possess any typical transmembrane domain, it may adhere peripherally to the plasma membrane via its affinity to SGG. Our preliminary results have shown that AS-A conjugated with Alexa-430 binds in a concentration dependent manner to SGC (SGG's analog) immobilized in microtiter plate wells. Since the SGG desulfated product, galactosylglycerolipid (GG), is not detected in sperm, it is unlikely that the putative AS-A-SGG interaction would be through the enzyme's active site pocket. We suggest that SGG may bind to the exposed basic amino acids of AS-A, such as R19, R114, R214 and R217. The availability of the active site pocket would allow surface AS-A on live sperm to desulfate NCS as described above. This work is supported by CIHR grant MT10366.
KEY WORDS: arylsulfatase A, sperm surface, enzymatic properties, sulfoglycolipid