SASD

Reported applications of SASD involve modification of lipopolysaccharide (LPS) molecules and studying their interaction with albumin and an antibody directed against LPS (Wollenweber and Morrison, 1985), identification of the murine interleukin-3 receptor and an N-formyl peptide receptor (Sorenson et al., 1986), crosslinking of factor V and Va to iodinated peptides 

The best radiolabeling technique for SASD is to use the Iodogen method (Shephard et al., 1988) described in Chapter 12, Section 3. The following suggested protocol for using SASD was based on the method described in the Thermo Fisher Catalog. 

The following operations should be done using standard safety procedures for working with radioactive compounds. All steps involving SASD prior to initiation of the photoreaction should be done protected from light to avoid loss of phenyl azide activity. The radiolabeling procedure should be done quickly to prevent excessive loss of NHS ester activity due to hydrolysis. 

Radiolabel 55 nmol of SASD using IODO-GEN (Thermo Fisher) and 40 pCi Na 125I for 30 seconds. Do not use chloramine-T, since termination of the iodination reaction with this reagent involves addition of a reducing agent which may cleave the disulfide bonds of the crosslinker. 

React for 30 minutes to create the SASD derivative, coupled through the NHS ester-reactive group of the crosslinker onto available amine groups of the protein (forming amide bonds). 

Rgure 173 The hydroxyl group on the phenyl azide ring of SASD may be iodinated with to allow radiolabeling studies to be done on photolyzed conjugates. 

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Figure 5.18 SASD is a photoreactive crosslinker that can be used to modify amine-containing compounds through its NHS ester end and subsequently photoactivated to initiate coupling with nucleophiles (after ring expansion to an intermediate dehydroazepine derivative). The crosslinks may be selectively cleaved at the internal disulfide group using DTT.

Chattopadhyay et al., 1992), and a comparison of radiolabeling techniques for the crosslinker (Shephard et al., 1988). Other studies have involved the investigation of protein interactions using the label transfer nature of radioiodinated SASD (Gupta et al., 2005 Lindersson et al., 2005 LeFebvre et al., 2006). 

Add the SASD-modified protein to a second protein or other molecule to be conjugated. After mixing, expose the solution to long-wave UV light for 10-15 minutes at room temperature to effect the conjugation. The solution may be kept on ice to prevent over-heating of sensitive proteins. 

Iodination of the crosslinker may be done according to the procedures discussed in Chapter 12, Section 5, or performed similar to that described for SASD (Section 3.2, this chapter). 

SASD (sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-l,3 -dithiopropionate) (Chapter 5, Section 3.2) behaves in a similar manner, except it contains an amine-reactive end that can be... 

SASD free sialic acid storage disease... 

Three different rare genetic metabolic defects in sialic acid metabolism are known, as indicated in Fig. 4.3.2 [3, 21] (1) free sialic acid storage disease (SASD Online Mendelian Inheritance in Man, OMIM 604369, 269920), a lysosomal membrane transporter defect (2) sialuria (OMIM 269921), a feedback inhibition defect in sialic acid biosynthesis (3) sialidosis (OMIM 256550), a breakdown defect of sialyloli-gosaccharides caused by a defect of lysosomal sialidase. In all these genetic defects, an increased amount of sialic acid can be found in tissues and or body fluids, either bound to OGSs as in (3), or in its free state as in (1) and (2). 

Fig. 4.3.2 Human sialic acid metabolism and genetic defects. -6P -6-Phosphate, -9P -9-phos-phate, CMP cytidine 5 -monophosphate, CTP cytidine 5 -triphosphate, UDP-GlcNAc uridine diphosphate-N-acetyl-D-glucosamine, ManNAc N-acetylmannosamine, NeuAc N-acetylneur-aminic acid, OGS oligosaccharides, SASD sialic acid storage disease...

The lysosomal disorder SASD is characterized by accumulation of the free acid monosaccharide sialic acid in the lysosomal compartment of the cell. Diagnosis is based on the demonstration of abnormal excretion of free, not OGS-bound sialic acid in urine, coupled with accumulation of free sialic acid in cultured fibroblasts, and on microscopic evidence of vacuoles (increased and swollen lysosomes filled with light electron-lucent material in skin biopsy and peripheral blood lymphocytes). The inheritance is autosomal recessive. There are different clinical forms of this disorder an adult form, called Salla disease (SD) or Finnish sialuria (OMIM 604369) infantile SASD (ISSD OMIM 269920) and an intermediate form, severe Salla disease [3,16]. 

SASD must be discriminated from other disorders of sialic acid storage [3] (1) sialidosis and galactosialidosis, defects respectively in lysosomal sialidase and both sialidase and /1-galactosidase. (OMIM 256550 and 256540) (2) nonlysosomal sialuria (OMIM 269921). 

Table 4.3.1 Disorders of sialic acid metabolism with storage of sialic acid, clinical, and biochemical discrimination. ISSD Infantile sialic acid storage disease, OGS oligosaccharide, SASD free sialic acid storage disease...

Free sialic acid storage disorders, SASD ... 

Table 4.3.4 Some typical pathological free NeuAc values in urine samples of different clinical forms of SASD (values from own experience and the literature) ... 

The test is performed for diagnosis of all clinical forms of SASD. This analysis is usually done after an initial TLC screening test that is positive for free sialic acid, and an increased free sialic acid value in the quantitative urine determination test. The test is like the quantitative urine test performed with the periodate-TBA assay [5, 22]. However, in this case interference is decreased by prepurification of the sample using ion-exchange chromatography [12]. Fibroblasts are cultured under standardized conditions. Cell lysates are prepared by tip sonification in distilled water and the cleared lysates are applied to small Dowex columns. NeuAc is eluted, freeze dried,... 

This method may serve in the future as the reference method for free sialic acid in the diagnosis of SASD. 

Verheijen FW, Mancini GM (2003) Lysosomal sialic acid transporter sialin (SLC17A5) sialic acid storage disease (SASD). In Broer S, Wagner CA (eds) Membrane Transporter Diseases. Kluwer Academic/Plenum, New York, pp 233-239... 

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