SELECTIVE DEAMIDATION OF RECOMBINANT HUMAN STEM-CELL FACTOR DURING IN-VITRO AGING - ISOLATION AND CHARACTERIZATION OF THE ASPARTYL AND ISOASPARTYL HOMODIMERS AND HETERODIMERS
Yr. Hsu et al., SELECTIVE DEAMIDATION OF RECOMBINANT HUMAN STEM-CELL FACTOR DURING IN-VITRO AGING - ISOLATION AND CHARACTERIZATION OF THE ASPARTYL AND ISOASPARTYL HOMODIMERS AND HETERODIMERS, Biochemistry, 37(8), 1998, pp. 2251-2262
During in vitro aging, deamidation of recombinant human stem cell fact
or produced in Escherichia. coli was detected by HPLC analysis and by
the release of soluble ammonia, The deamidation rate is very slow in b
uffers at low pH or at low temperatures; however, the rate is signific
antly accelerated in alkaline buffers such as sodium bicarbonate in co
mbination with elevated temperatures, HPLC isolation of various deamid
ated forms followed by peptide mapping and mass spectrometric analyses
revealed that the deamidation involves Asn(10) in the sequence -(TNNV
)-N-9- near the N-terminus of the protein. Following peptide mapping a
nalysis, significant amounts of aspartyl and isoaspartyl peptides were
identified, indicating the conversion of asparagine into both asparta
te and isoaspartate residues. As a result of spontaneous association-d
issociation of stem cell factor dimer, a total of five deamidated form
s, including two homodimers and three heterodimers, were detected and
isolated. Cell proliferation assays showed that two rhSCF heterodimeri
c species, derived from dimerization between isoaspartyl and other ste
m cell factor monomers, retain only approximately half of the biologic
al activity. The homodimer with isoaspartic acid in place of Asn(10) i
s 50-fold less potent, while the aspartyl homodimer, either isolated d
uring deamidation experiments or recombinantly prepared by site-direct
ed mutagenesis (e.g., N10D and N10D/N11D variants), exhibits higher ac
tivity than the standard molecule. In comparison, synthetic N10A and N
10E variants, though missing the deamidation site, are significantly l
ess active. All these variants lacking the Asn(10) deamidation site ar
e relatively more stable than those containing the asparagine residue.
The results indicate that the biological function and chemical stabil
ity of stem cell factor are influenced by the nature of the residue at
position 10.