Human Sult1a2

A human SULT1A2 cDNA (ST1A2) was first cloned from a human liver library by Ozawa et al. (1995). Our laboratory subsequently cloned two cDNA forms of this enzyme, HAST4 and HAST4v, that differed by two amino acids (Thr7 to Ile and Thr235 to Asn). The coding region of HAST4 and HAST4v are 97% and 94%

homologous to SULT1A1 and SULT1A3, respectively (Zhu et al., 1996). Interestingly, when both forms are expressed in COS cells, they exhibit markedly different affinities for p-nitrophenol, with Km values of 74 ^M and 8 ^M for HAST4 and HAST4v, respectively. For the same reaction, SULT1A1 and SULT1A3 exhibit Km values of 0.7 and 2200 ^M, respectively (Zhu et al., 1996). The effect of a change from Asn235 to Thr in SULT1A2 was investigated using the crystal structure of SULT1A1 (Gamage et al., 2003). Asn235 is a highly conserved residue in the SULT1A family. This residue is located at the end of helix a 12 and at the beginning of the large flexible loop that undergoes disorder-order transition upon substrate binding (Figure 10.3; see color photo insert following p. 210). The side chain oxygen of this residue forms a hydrogen bond with the main chain nitrogen of Met237, and this interaction could be important for maintaining the stability of the flexible loop interactions (Thr235 does not form this interaction). It is noteworthy that the Phe247, which interacts with both p-nitrophenol molecules is located in this flexible loop region. Based on this structural observation, we propose that disruption to flexible loop interactions could affect the enzyme's stability.

In a more recent human population study of DNA isolated from human liver tissue obtained from 61 Caucasian patients, Raftogianis et al. (1999) reported 13 different allelic variants of SULT1A2 that encode four different amino acid changes, which resulted in six different SULT1A2 allozymes (Table 10.5). These changes

SUTiTlAl SULT1A2 SULT1A3

SULT1A1 51 3ULT1A2 51 SULT1X3 51

SULT1A1 101; SULT1A2 101: SULT1AJ 101 :

SULT1A1 151 : 3ULT1A2 151; SVLT1A3 151:

SULT1A1 201: SULT1A2 201: SULT1A3 201;

SULT1A1 251: SULT1A2 251: 3ULT1A3 251:

S3 PS8

meliqdtsrp pleïvkgvpl ikïfaealgp lqsfqarpdd lliètïpksc meliqdtsrp pleïvkgvpl ikïfaealgp lqsfqarpdd ll1stïpksc meliqdtsrp pleïvkgvpl IkïFAEALGP lqsfqarpdd LLIhTÏ'PKSi a2

itwvsqildm iïqggdlekc hrapifmrvf flefkapgip sgmetlkdtf irwvsqildm iïqggdlekc hrapifmrvp fî.efkvpgip êgmetlkntp irwvsqildm iïqggdlekc nrapiyvrvp flevndpgep sgletlkdtp

^^m mm aprllkthlp lallpqtlld qkvkwyvafe aprllkthlp lallpqtlld qkvkwïvar pprlikshlp lallpqtlld qkvkvvyvar

3'PB

nakdvav nakdvav npkdvav a?

epgtwdsfle kfmvgevsyg swyqhvqeww elsrthpvly lfïedmkenp hpgtwesfle kfmagevsïg swyqhvqeww elsrthpvly lfïedmkenp epgtwdsfle kfmagevsïg swyqhvqeww elsrthpvly lfïedmkenp a!0

ail a]2

kreiqkilef vghslpeetv dfmvqhtsfk emkknpmtny ttvfqefmdh kreiqkilef vgrslpeetv dlmvehtsfk emkktpmtny ttvrrefhdh kreiqkilef vgrslpeetm dfmvqhtsfk emkknfmtnï ttvpqelmdh

3'PB

sïspf sispf

SISPF

agdwkttftv aqnerfdady aekmagcsls frsel agdwkttftv aqnerfdady aekmagcsls frsel agdwkttftv aqnerfdady aekmagcsls frsel

50 50 50

100 100 100

150 150 150

200 200 200

250 250 250

295 295 295

figure 10.3 Sequence alignment of human SULT1A1, 1A2, and 1A3. Secondary structure elements are numbered based on SULT1A1 structure. Residues conserved in PSB loop and 3'PB site are boxed, and those that differ among the isoforms are shown in red. Residues that line the substrate-binding pocket are highlighted in yellow (based on SULT1A1 structure). (See color photo insert following p. 210.)

TABLE 10.4

Frequency of the SULT1A1 *1 and SULT1A1 *2 (R213H) Polymorphisms in Different Ethnic Groups

Allele Frequency

TABLE 10.4

Frequency of the SULT1A1 *1 and SULT1A1 *2 (R213H) Polymorphisms in Different Ethnic Groups

Allele Frequency

Population

R213H

Mean Age

Female:Male

Reference

Ethnicity

Wildtype

Mutant

#

(years)

Ratio

Caucasian

0.69

0.31

150

Raftogianis et al.,

1997

Caucasian

0.68

0.32

293

53.6

0.56

Coughtrie et al.,

African

0.73

0.27

52

1999

Japanese

0.83

0.17

143

61

0.81

Ozawa et al., 1999

Caucasian

0.66

0.34

150

40-64

Male

Engelke et al.,

2000

Caucasian

0.61

0.39

189

1.27

Steiner et al.,

Caucasian with

0.65

0.35

134

2000

prostate cancer

Caucasian

0.659

0.341

156

Female

Caucasian with

0.584

0.416

332

Female

Zheng et al., 2001

breast cancer

Caucasian

0.64

0.36

211

0.66

Nowell et al.,

African

0.74

0.26

40

0.66

2000

Caucasian

0.66

0.33

242

Carlini et al., 2001

African-American

0.48

0.29

70

Chinese

0.91

0.08

290

Female

Caucasian

0.68

0.32

402

71.27

0.66

Wong et al., 2002

Caucasian with

0.67

0.33

383

68.36

0.77

colorectal cancer

(Hsa) SULT1A2 Allozymes

Amino Acid Frequency

TABLE 10.5

(Hsa) SULT1A2 Allozymes

Amino Acid Frequency

SULT1A2 Allozyme

7

19

184

235

(Caucasian Liver Samples*)

Reference

1A2*1 (wildtype)

Ile

Pro

Arg

Asn

0.51

Zhu et al., 1996 Raftogianis et al., 1999

1A2*2

Thr

Pro

Arg

Thr

0.29

Zhu et al., 1996 Raftogianis et al., 1999

1A2*3

Ile

Leu

Arg

Asn

0.18

Ozawa et al., 1995 Raftogianis et al., 1999

1A2*4

Thr

Pro

Cys

Thr

0.008

Raftogianis et al., 1999

1A2*5

Thr

Pro

Arg

Asn

0.008

Raftogianis et al., 1999

1A2*6

Ile

Pro

Arg

Thr

0.008

Raftogianis et al., 1999

resulted in >60-fold variation in the Km for p-nitrophenol, 10-fold variation in the Km for PAPS, and ~50-fold variation in the IC50 for DCNP on p-nitrophenol sul-fonation by these allozymes. Population studies have shown that ethnic differences exist in the expression of these enzymes. Carlini et al. (2001) showed that SULT1A2*1,1A2*2, and 1A2*3 were present at frequencies of 0.51, 0.39, and 0.10, respectively, in the Caucasian population, whereas in the Chinese population the frequencies were 0.92 for SULT1A2*1 and 0.08 for SULT1A2*2 and the 1A2*3 allele was not detectable. In the African-American population the frequencies of SULT1A2*1, 1A2*2, and 1A2*3 were 0.64, 0.25, and 0.11 respectively (Carlini et al., 2001). In a model Salmonella typhimurium system, Meinl et al. (2002) have shown that these polymorphic forms of SULT1A2 have different activities toward certain aromatic amines and amides, which may affect individual susceptibility to these carcinogens. The most striking functional difference between this new SULT1A member, SULT1A2, and SULT1A1 and SULT1A3 is that it exhibited no activity toward dopamine as a substrate (Zhu et al., 1996). Ozawa et al. (1995) have shown that COS cell expressed SULT1A2 (ST1A2) to sulfonate p-nitrophenol, minoxidil, and P-naphthol at significantly lower rates than SULT1A1 (ST1A3). The same authors also reported a large difference in Km values for p-nitrophenol sulfona-tion between SULT1A2 and SULT1A1: 20 ^M for SULT1A2 and 1.4 ^M for SULT1A1. Most studies have focused on the SULT1A1 and SULT1A3 enzymes, as the validity of SULT1A2 as a functionally significant enzyme is still questionable. Little data exist on the tissue distribution of this enzyme, and mRNA profiles suggest lower expression levels in most tissues and cell lines tested compared to SULT1A1 and SULT1A3 (Dooley et al., 2000). These reverse transcription PCR (RT-PCR) studies also suggest the presence of a SULT1A2 mRNA species that is incorrectly spliced, to contain an intron. This may result in at least some portion of the SULT1A2 mRNA never being translated into a functional protein (Dooley et al., 2000). While Figure 10.2 shows that SULT1A2 protein is not detectable in the ten human liver cytosols tested, it nonetheless is interesting to note that Ozawa et al. (1995) were able to show an approximate twofold difference in the levels of both SULT1A2 (ST1A2) and SULT1A1 (ST1A3) mRNA levels in livers from six human subjects.

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