C. Yeang et aL/ Biochimica et Biophysica Acta 1781(2008)610-617 615 As shown in Fig. 3B, a portion of SMS2-Flag(WT)was located on 3. 2. Analysis of a domain unique to SMSI on subcellula plasma membrane where it co-localized with cadherin(a well-know plasma membrane marker), and a portion was found in the peri- ASl is a Golgi protein which has been co-localized with the golgi nuclear region where it co-localized with Golgi marker a Mannosi- marker Mannosidase ll [17](Fig 2B). SMS2 on the other hand localizes dase ll. This result also confirmed a previous report[17. Furthermore, to both the plasma membrane as well as the Golgi [17 ( Fig. 3B). There all mutants have an identical cellular distribution as WT (Fig. 3B and are no known or predicted targeting signals in either protein. C), suggesting that these point mutations only influenced SMS2 Although the genes encoding these two isoforms are located on catalytic activity but not the enzyme topology distinct chromosomes, SMSI and SMS2 are 51.5% identical in protein So far, no experimental studies identifying the locale of an SMs sequence active site have been reported. In this study, we utilized site- Sequence-wise, the most striking difference between the two directed mutagenesis to elucidate the catalytic structure for both isoforms is that SMSI, but not SMS2 has an N-terminal Sterile alpha SMSI and SMSZ SMS activity is closely related with four important Motif (SAM) domain. We explored the possibility that this SMs1 lipids, ceramide, DAG, SM, and phosphatidylcholine, which are unique sequence may play a role in the differential subcellular involved in plasma membrane formation, signal transduction, and localization of SMSI and SMS2 lipoprotein metabolism. The catalytically inactive enzymes created Truncation of 61 amino acids from the sms1 sam domain resulted by this study will be ful in the future studies in a mutant with an identical distribution pattern as the wild type SMS is a therapeutie for the treatment of disease enzyme( Fig. 5C). Furthermore, addition of these 61 amino acids to the cancer and atheros and elucidating the amino N-terminus of SMS2 did not alter localization of the protein( Fig. 5D). form an active site will provide a molecular basis for de- This was surprising because trans-Golgi resident proteins have been signing the drugs known to target to the Golgi through aggregation and subsequent Based on the LPP structure [18 and through homology modeling, exclusion from transport vesicles. SAM, conventionally thought of as a we have created a tertiary structure model of SMS1, which shows a protein interaction domain, has also been shown to mediate homo- favorable distance of 4.27 A for the interaction between H328 and oligomerization [21 Although the results are not expected, we D332, as well as an 11.90 A distance between H328 and H283 which conclude that the SMS1 SAM domain is neither necessary nor can accommodate the phosphate group from phosphatidylcholine sufficient for Golgi targeting of SMS. The domains, responsible to (Fig 4). This model is also applicable to SMSZ From this model, we can subcellular distribution of both enzymes, should be located within the clearly see that the replacement of any one of these three amino acids region where SMSI and SMS2 share the similarity. Furthermore, the abolishes SMSI or SMS2 activity in the cells. SAM domain does not influence SMS activity As shown in Fig 5B, 1190A otein(PDB entry 2IC8)and PhoN protein(PDB entry 2AKC), with known structures, were used as templates. Multiple sequence alignments and construction of homology models the respective distances between residues are depicted.As shown in Fig. 3B, a portion of SMS2-Flag (WT) was located on plasma membrane where it co-localized with cadherin (a well-known plasma membrane marker), and a portion was found in the peri￾nuclear region where it co-localized with Golgi marker α Mannosi￾dase II. This result also confirmed a previous report [17]. Furthermore, all mutants have an identical cellular distribution as WT (Fig. 3B and C), suggesting that these point mutations only influenced SMS2 catalytic activity but not the enzyme topology. So far, no experimental studies identifying the locale of an SMS active site have been reported. In this study, we utilized site￾directed mutagenesis to elucidate the catalytic structure for both SMS1 and SMS2. SMS activity is closely related with four important lipids, ceramide, DAG, SM, and phosphatidylcholine, which are involved in plasma membrane formation, signal transduction, and lipoprotein metabolism. The catalytically inactive enzymes created by this study will be very useful in the future studies. Potentially, SMS is a therapeutic target for the treatment of diseases, such as cancer and atherosclerosis, and elucidating the amino acids that form an active site for SMS will provide a molecular basis for de￾signing the drugs. Based on the LPP structure [18] and through homology modeling, we have created a tertiary structure model of SMS1, which shows a favorable distance of 4.27 Å for the interaction between H328 and D332, as well as an 11.90 Å distance between H328 and H283 which can accommodate the phosphate group from phosphatidylcholine (Fig. 4). This model is also applicable to SMS2. From this model, we can clearly see that the replacement of any one of these three amino acids abolishes SMS1 or SMS2 activity in the cells. 3.2. Analysis of a domain unique to SMS1 on subcellular localization SMS1 is a Golgi protein which has been co-localized with the Golgi marker Mannosidase II [17] (Fig. 2B). SMS2 on the other hand localizes to both the plasma membrane as well as the Golgi [17] (Fig. 3B). There are no known or predicted targeting signals in either protein. Although the genes encoding these two isoforms are located on distinct chromosomes, SMS1 and SMS2 are 51.5% identical in protein sequence. Sequence-wise, the most striking difference between the two isoforms is that SMS1, but not SMS2 has an N-terminal Sterile Alpha Motif (SAM) domain. We explored the possibility that this SMS1 unique sequence may play a role in the differential subcellular localization of SMS1 and SMS2. Truncation of 61 amino acids from the SMS1 SAM domain resulted in a mutant with an identical distribution pattern as the wild type enzyme (Fig. 5C). Furthermore, addition of these 61 amino acids to the N-terminus of SMS2 did not alter localization of the protein (Fig. 5D). This was surprising because trans-Golgi resident proteins have been known to target to the Golgi through aggregation and subsequent exclusion from transport vesicles. SAM, conventionally thought of as a protein interaction domain, has also been shown to mediate homo￾oligomerization [21]. Although the results are not expected, we conclude that the SMS1 SAM domain is neither necessary nor sufficient for Golgi targeting of SMS. The domains, responsible to subcellular distribution of both enzymes, should be located within the region where SMS1 and SMS2 share the similarity. Furthermore, the SAM domain does not influence SMS activity. As shown in Fig. 5B, Fig. 4. A favorable conformation of active site of human SMS1. The three-dimensional structural model of human SMS1 was built through homology modeling. Two proteins, GlpG protein (PDB entry 2IC8) and PhoN protein (PDB entry 2AKC), with known structures, were used as templates. Multiple sequence alignments and construction of homology models were conducted by using the Modeler module in the Discovery Studio software (version 1.6, Accelrys Inc.). The conserved histidine 285, histidine 328, and histidine 332 along with the respective distances between residues are depicted. C. Yeang et al. / Biochimica et Biophysica Acta 1781 (2008) 610–617 615