Leiden Muscular Dystrophy pages

Sarcospan (SSPN)

(last modified October 3, 2003)



Contents




Summary


Sarcospan (SSPN, Crosbie), originally described as 25DAP / A5 (Ervasti, Yoshida), was the last protein of the DGC that could be characterized at the molecular level. Cloning of the gene was hampered mainly by the difficulty to raise antibodies against the purified protein (rabbit protein injected in sheep and goat). Crosbie et al. purified 25DAP / A5 from rabbit skeletal muscle, micro-sequenced two Lys-C digestion products and detected a database homology with one peptide of the human and mouse Kirsten ras-associated gene (KRAG). The sarcospan gene maps to chromosome 12p11.2, overlapping the locus for CFEOM (congenital fibrosis of the extraocluar muscle) locus, mapped at 12p11.2-q12. Sarcospan encodes a major 6.5 kb transcript present exclusively in skeletal and cardiac muscle, and a 4.5 kb transcript in many other tissues. The primary sarcospan amino acid sequence predicts four transmembrane spanning domains and consensus phosphorylation sites for cAMP-dependent protein kinase, protein kinase C and casein kinase II. Overall sarcospan topology is similar to that of the tetraspan proteins.


The sarcospan gene


The human sarcospan gene (SSPN) was cloned by ... using ... cDNA. The SSPN gene maps to human chromosome 12p11.2 (Heighway J., Genomics 35: 207).

Exon Exon size (bp) Intron size (kb) 5' cDNA position Splice after Remarks
1         5'UTR / .. bp coding

Legend:
Exon: numbering of exons and intron/exon boundaries are according to...with the first base of the Met-codon counted as position 1 (see Reference sequence). Exon size: size of exon indicated in basepairs. Intron size: size of intron indicated in kilobasepairs. 5' cDNA position: first base of the exon (according to cDNA sequence reported by ...). Splice after: splicing occurs in between of two coding triplets (0), after the first (1) or the second (2) base of a triplet. Remarks: 5'UTR = 5' untranslated region, 3'UTR = 3' untranslated region.

primers for DNA-amplification


The empty mRNA


On Northern blots, sarcospan RNA expression was detected as a 6.5 kb transcript present exclusively in skeletal and cardiac muscle (Crosbie). A 4.5 kb transcript was detected in skeletal and cardiac muscle, colon, ovary, prostate, small intestine, spleen, testis and thymus.

primers for RNA-amplification


The sarcospan protein


Crosbie et al. purified 25DAP / A5 from rabbit skeletal muscle and micro-sequenced two Lys-C digestion products (I: KHRYQVFYVGV and II: KDRQPRGQQRQGDAAGPDDPG) and detected a database homology of peptide I with the human and mouse Kirsten ras-associated gene (KRAG). The murine KRAG gene was first identified by co-amplification of its transcript with Kirsten ras in adrenal carcinoma cells (...), while human KRAG was identified through analysis of KRAS2 co-amplified transcripts in lung and ovarian carcinoma cells (...).

The primary sarcospan amino acid sequence predicts four transmembrane spanning domains (amino acids Leu54-Val83, Ala90-Val107, Met121-Phe142 and Val187-Trp217) and consensus phosphorylation sites for cAMP-dependent protein kinase (Thr33), protein kinase C (Ser120 and Thr190) and casein kinase II (Thr157 and Thr234). Overall sarcospan topology is similar to that of the tetraspan proteins. Sarcospan's name was derived from its membrane topology and its presence in the sarcolemma (Crosbie). Sarcospan differs from KRAG by having an additional 26 amino acids at its N-terminus. To account for these differences, Crosbie redesignated KRAG to sarcospan-1 (SPN1) and the newly identified protein to sarcospan-2 (SPN2). Evolutionary, sarcospan shows closest relation to ROM-1, peripherin and uroplakin.

The tetraspans or transmembrane 4 superfamily (TM4SF) and the tetraspanins have 4 conserved transmembrane spanning alpha-helices. TM4SF memebrs have no clear function, but many members are associated with integrins and implicated in the regulation of cell proliferation (...). Sarcospan is the only member of the dystrophin-associated glyco-protein complex (DGC) which spans the membrane more than once, holding it firmly in the lipid bilayer and thereby anchoring the DGC. The multiple transmembrane regions might form a pore in the sarcolemma, serving as a membrane channel. The charged residues in the TM-domains might be important for protein-protein interactions, possibly with integrins or components of the DGC.

Sarcospan antibodies

Crosbie describes a rabbit polyclonal antibody, raised by injecting a synthetic N-terminal peptide (CAA-DRQPRGQQRQGDAAGPD). Affinity-purified antibody did not react with mouse tissue but does stain a 25 kDa protein on Westernblot in DGC purified from rabbit skeletal muscle membranes. Immunohistochemical analysis of human skeletal muscle sections shows a clear sarcospan membrane staining. However, sarcospan staining  is dramatically reduced in muscle from Duchenne patients (Crosbie).


Sarcospan and disease


No link between sarcospan and disease has been reported. However, an overlap between the CFEOM (congenital fibrosis of the extraocluar muscle) locus, mapped at 12p11.2-q12 in two large unrelated families (Engle EC, Am.J.Hum.Genet. 57: 1086) and the SSPN gene (12p11.2) does exist.


Sarcospan polymorphisms


Exon Polymorphism RE-site Protein Reference DNA/RNA Technique Frequency
               

Legend: Polymorphisms are described basically as suggested by the AHCMN and Beutler et al., with minor deviations/additions (see Nomenclature).


Miscellaneous


DNA-primers

Amplified Length Reference forward primer reverse primer Name
           
           
           

Legend:
Exonic sequences are in upper case, intronic and gene flanking sequences in lower case and added primer tails in italics. Amplified: region amplified. Numbering of exons is according to... Length: length of PCR-product in basepairs. Reference: publication describing the primer(s). Forward primer: sequence of forward primer. Reverse primer: sequence of reverse primer. Name: name of the primers.


RNA-primers

Amplified Length Reference Forward primer Reverse primer Name
           
           
           

Legend:
Exonic sequences are in upper case, intronic and gene flanking sequences in lower case and added primer tails in italics. Amplified: region amplified. Numbering of exons is according to.... Length: length of PCR-product in basepairs. Reference: publication describing the primer(s). Forward primer: sequence of forward primer. Reverse primer: sequence of reverse primer. Name: name of the primers.


Sarcospan sequences



| sarcoglycanopathies |
| Top of page | Sarcoglycan homepage | LMDp homepage |
| Remarks / information | Disclaimer |