DPAGT1 Myasthenia and Myopathy. Genetic, Phenotypic, and Expression Studies

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Supplementary Data

DPAGT1 myasthenia and myopathy. Genetic, phenotypic, and expression studies.

Duygu Selcen, MD, Xin-Ming Shen, PhD, Joan Brengman, BS, Ying Li, PhD, Anthony A. Stans, MD, Eric Wieben, PhD,and Andrew G. Engel, MD

Contents

Appendix e-1, e-2, and e-3

Figure e-1

Figure e-2

Appendix e-1

Real-Time Quantitative PCR and RNA Splicing Studies. mRNA was extracted from muscle with the Micro-FastTrack 2.0 mRNA isolation kit (Life Technologies). We employed allele-specific real-time quantitative PCR to assess expression of the transcript with skipped exons 2 and 3. Reactions were performed in triplicate, and gene expression values were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

For RNA splicing experiments we cloned genomic DNA of Pt 2 containing exons 2 to 4 with their flanking intronic regions into Exontrap vector pET according to the manufacturer’s instructions (Boca Scientific, Boca Raton, FL). pET vectors harboring wild type and mutant DNA variants were transfected into COS-7 cells (monkey kidney fibroblasts). Forty-eight hours after transfection, mRNA was extracted from the cells and reverse transcribed into cDNA with Micro-FastTrack 2.0 mRNA isolation kit and SuperScript VILO cDNA synthesis kit (Life Technologies). cDNA was amplified using vector-specific primers, run on 1% agarose-TAE (Tris-Acetic Acid-EDTA) gel, visualized under UV/ethidium bromide and the bands from the gel were purified and sequenced.

Appendix e-2

Assay of DPAGT1 Enzyme Activity Full-length cDNAs containing missense variants and control cDNA were cloned into pCMV-Myc-C vector (Clontech, Mountain View, CA).COS-7 cells were transfected with wild type or mutant DPAGT1 using TransIT-LT1 reagent (Mirus, Madison, WI). The enzyme assay was based on that described by Wallis1 in presence of 13 μM dolichol-phosphate (generous gift from Dr. Hudson Freeze), modified by addition of phosphatidyl glycerol for stabilizing the enzyme,2 and by protease and phosphatase inhibitors and antioxidants. In each assay, the activity in transfected cells was corrected for endogenous activity of untransfected cells and for the level of expression of the analyzed proteins determined by immunoblotting.Twenty-four hours after transfection, transfected and nontransfected cells were pelleted and resuspended in a solution containing 50 mM TrisCl, pH7.5, 200 mM KCl, 10 mM MgCl2, 5mM AMP, 10 mM dithiothreitol, 0.8%, 1 μg/ml leupeptin,1μg/ml of pepstatin, 5 mM 2-mercaptoethanol, 0.8%TritonX-100, 0.25% NonIdet (Solution A) plus 100 μg/ml DNAse1. All subsequent procedures were carried out at 2-4˚C. The pellet was washed with TBS containing 5 mM EDTA, resuspended in 1 ml TBS and aliquots were taken for cell count, protein measurement with the BCA reagent (Thermo Fisher Scientific, Rockford, IL). Thecellswerelysedbysonicationinanicebathbythree20secpulsesandby freezing andthawingthreetimes.Thefinalreactionmixtureconsistedof150μlSolutionA plus1.3mMphosphatidylglycerol,2mMorthovanadate,13μMUDP-[16-3H]GlcNAc(860dpm/pmol),13μMdolichol-phosphateandalysateof~1millioncells.Both UDP-GlcNAc and dolichol phosphate were present in several-fold molar excess over the respective apparent Kms of the enzyme.3Afterpreincubationfor10minat30˚C,thereactionwasstartedbyaddingthecelllysate.Thereactionwasterminatedafter10minwith1mlofa3:2v/vmixtureofice-coldchloroform-methanol.Aftercentrifugationat1500xgfor10min,thechloroformlayerwasaspiratedandfurtherpurifiedwith3washesof4mMMgCl2inwater,transferredtoascintillationvial,evaporatedtodrynessunderastreamofN2,andcounted24hoursafteradditionof10mlUltimaGold(Perkin–Elmer,Waltham,MA). In each assay, the activity in transfected cells was corrected for endogenous activity of untransfected cells and for level of expression of the analyzed proteins determined by immunoblotting.

Reference

(1) Wallis GLF, Hemming FW, Peberdy JF. Investigation of the glycosyltransferase enzymes in the initial stages of the N-linked protein glycosylation pathway in Aspergillus niger. Biochim Biophys Acta 1999;1426:91-98.

(2) Kaushal GP, Elbein AD. Purification and properties of UDP-GlcNAc:dolichyl-phosphate Gl-NAc-1-phosphate transferase. Activation and inhibition of the enzyme. J Biol Chem 1985;260:16303-16309.

(3) Wu X, Rush JS, Karaoglu D, et al. Deficiency of UDPG-GlcNAc:dolichol phoshate N-acetylglucosamine-1 phosphate transferase (DPAGT1) causes a novel congenital disorder of glycosylation type Ij. Hum Mutation 2003;22:144-150.

Appendix e-3

Structural Studies.

Cryosections were used to colocalize the acetylcholine receptor (AChR) with rhodamine labeled -bungarotoxin (-bgt) and acetylcholine esterase (AChE) with a monoclonal anti-AChE antibody.1 AChE was also visualized on teased, glutaraldehyde-fixed muscle fibers cytochemically.2 EPs were localized for electron microscopy3 and quantitatively analyzed4 by established methods. Peroxidase-labeled -bgt was used for the ultrastructural localization of AChR.5 The number of AChRs per EP was measured with [125I]bgt.6

Reference List

(1) Fambrough DM, Engel AG, Rosenberry TL. Acetylcholinesterase of human erythrocytes and neuromuscular junctions: Homologies revealed by monoclonal antibodies. Proc Natl Acad Sci USA 1982;79:1078-1082.

(2) Gautron J. Cytochimie ultrastructurale des acétylcholinestérases. Microscopie 1974;21:259-264.

(3) Engel AG. The muscle biopsy. In: Engel AG, Franzini-Armstrong C, eds. Myology, 3rd ed. New York: McGraw-Hill, 2004:681-690.

(4) Engel AG. Quantitative morphological studies of muscle. In: Engel AG, Franzini-Armstrong C, eds. Myology, 2nd ed. New York: McGraw-Hill, 1994:1018-1045.

(5) Engel AG, Lindstrom JM, Lambert EH, Lennon VA. Ultrastructural localization of the acetylcholine receptor in myasthenia gravis and in its experimental autoimmune model. Neurology 1977;27:307-315.

(6) Engel AG. The investigation of congenital myasthenic syndromes. Ann N Y Acad Sci 1993;681:425-434.

Figure e-1

Endplate region in patient 2. Note strong expression of AChR revealed by peroxidase labeled αbungarotoxin and abundant junctional sarcoplasm.

Figure e-2

Transferrin glycosylation patterns in control and patient sera. The upper panel shows isoelectric focusing of transferrin from two controls (C1 and C2) and Pt 1, Pt 2, and Pt 3. Numbers refer to sialic acid molecules attached to transferrin. M pattern displays all stages of glycosylation of transferrin. Pt 1 shows a normal pattern. In Pt 2 and Pt 3, the disialo band is more intense than trisialo band, and Pt 3 also has an asialotransferrin band. The lower panel displays capillary zone electrophoresis of serum transferrin from a healthy control and 3 patients. Numbers over peaks refer to sialic acid molecules attached to transferrin. Pt 1 has a normal pattern, whereas Pts 2 and 3 have a higher disialo- than trisialotransferrin peaks and Pt 3 again has a small asialotransferrin peak.