Characterization of functional domains of the cblD (MMADHC) gene product
Supplementary Material
Jehona Jusufi1, Terttu Suormala1, Patricie Burda1, Brian Fowler1, D. Sean Froese1*, Matthias R.
Baumgartner1,2,3*
1
Division for Metabolic Disorders and Children’s Research Center, University Children’s Hospital, Zurich,
Switzerland
2
radiz – Rare Disease Initiative Zurich, Clinical Research Priority Program for Rare Diseases, University of
Zurich, Switzerland
3
Zurich Center for Integrative Human Physiology, University of Zurich, Switzerland
*To whom correspondence may be addressed:
M.R.B.
Division for Metabolic Disorders and Children’s Research Center, University Children’s Hospital,
Steinweisstrasse 75, 8032 Zurich, Switzerland
Tel: +41 (0)44 266 7722
Fax: +41 (0)44 266 7167
Email: matthias.baumgartner@kispi.uzh.ch
D.S.F.
Division for Metabolic Disorders and Children’s Research Center, University Children’s Hospital,
Steinweisstrasse 75, 8032 Zurich, Switzerland
Tel: +41 (0)44 266 7305
Email : sean.froese@kispi.uzh.ch
Supplementary Methods
Transformed cblD-MMA/HC fibroblasts, grown in routine culture medium containing Dulbecco’s
Modified Eagle’s Medium (DMEM)(DMEM+GlutaMax™ ; Gibco), supplemented with 10% (v/v) foetal calf
serum (FCS; Gibco) and antibiotics (antibiotic-antimycotic mixture: penicillin 10'000 IU/ml, streptomycin
10'000 µg/ml, amphotericin B 25 µg/ml ) were used for transfections throughout the study. pTracerMMADHC-wt and mutant constructs were transiently transfected into these cells by electroporation. 25
µg vector DNA in a final volume of 30 µl (volume was compensated with sterile deionized water when
necessary) were pipetted into a 0.4 cm gene-pulser cuvette (BioRad) followed by 300 µl cell suspension
containing cells from one confluent T75cm2 culture flask in the above culture medium supplemented
with 1.25% dimethylsulfoxide (Sigma) (DMSO medium), incubated at room temperature for 1 min and
electroporated at 260V and 100 Ω with capacitance of 975 µF in a Gene Pulser II ( BioRad)
electroporator. Cells were divided between two T25cm2 culture flasks and grown in DMSO medium.
After 24 hours medium was removed, cell layers were rinsed once with 5 ml DMEM without any
supplements to remove traces of FCS, and each flask was covered with aluminium foil to protect the
cultures from light during the following incubation. To prepare incubation medium [57Co]-B12 Tracer
Stock in 0.9% benzylalcohol (57Co-cyanocobalamin; 10.5 µCi and 25 pg/ml; ICN Pharmaceuticals Inc) was
added to human serum (1 µl [57Co]-B12 Tracer Stock/ml serum) in a tube covered with aluminium foil and
preincubated for 30 min at 37°C to bind the labelled cobalamin to human transcobalamin. This serum
was then added to DMEM supplemented with antibiotics (1 ml serum/10 ml final medium), filtersterilized and 2 ml were added in each culture flask. Flasks were placed in cell incubator and cobalamin
coenzyme synthesis was estimated after 3 days incubation. Under illumination by photo-laboratory
grade red light only cells were harvested using trypsin, pelleted by centrifugation for 10 min at 1200 rpm
and resuspended in 5 ml phosphate buffered saline (PBS). For estimation of total uptake of 57Cocyanocobalamin (expressed as pg/mg protein) 1 ml of cell suspensions was added into a 1.5 ml
microcentrifuge tube, pelleted by centrifugation, radioactivity in pellet was quantitated in gamma
counter, and protein concentration estimated by the Lowry method. For extraction of cobalamin
derivatives the remaining 4 ml cell suspensions were centrifuged and pellets were spiked with 20 µl of a
solution containing unlabelled AdoCbl, MeCbl, CNCbl and OHCbl (each 1 mg/ml deionized water; Sigma).
Cells were lysed by 3 cycles of freezing on dry-ice (5 min) and thawing in a 37°C water bath. Extraction
of cobalamin derivatives was performed by addition of 1 ml absolute ethanol to the lysed cells with
incubation at 80°C in a heating block for 5-10min, centrifugation for 5 min at 1400 rpm, transfer
of the supernatant to a fresh tube, addition of a further 1 ml absolute ethanol and a repeat of
the procedure. The ethanol was then evaporated at 80°C in a heating block by flushing with
nitrogen gas. The extracted cobalamins were dissolved by letting them stand in 80 µl buffer A (0.05 M
phosphoric acid/ammonium pH 3.0) at room temperature for at least 15 min with 2-3 times Vortexmixing. Samples were transfered into microcentrifuge tubes and centrifuged for 5 min at 14000
rpm in a benchtop Eppendorf microcentrifuge to remove undissolved materials. The
supernatant was transferred into a new microcentrifuge tube and different cobalamin derivatives
were separated by HPLC (Jasco) from 50 µl of sample using a Lichrosorb RP-C8 column (Supelco) and
gradient elution at 25°C. The separation technique was modified from the original method (Jacobsen et
al. Anal Biochem 120: 394-403, 1982) to separate AdoCbl (retention time 25-27 min) from an unknown
radioactive compound eluting at 23-25 min. This was achieved by the following gradient elution, with
buffer A (0.05 M phosphoric acid/ammonium pH 3.0) and buffer B (buffer A supplemented with 30% v/v
acetonitrile): between 0 and 15 min after injection linear change from 100% A to 46% A + 54 % B which
was retained for 10 min; from 25 min to 26 min change to 100% B which was retained for 10 min;
between 36 min and 37 min change from 100% B to 100% A which was retained for at least 10 min
before the next injection. Fraction collection was started 15 min after injection and 48 fractions were
collecetd, each for 22 seconds, and counted. The retention times of hydroxocobalamin (OHCbl; 17-19
min), CNCbl (18-20 min), AdoCbl and MeCbl (30-31 min) were estimated by detecting unlabelled
compounds added to the cell pellet before extraction at 254 nm. The sum of counts per minute (cpm) in
fractions belonging to each cobalamin peak was calculated and the relative concentration of each
cobalamin compound was expressed as % of total cpm in all peaks (% of total cobalamins).
Each mutant constructs was transfected in at least 3 independent experiments. Transfections with an
empty pTracer vector, with MMADHC wildtype construct and with a construct containing one of the
missense mutant alleles detected in patients with cblD-HC defect (p.D246G, p.Y249C and p.L259P) were
included in each experiment.
Supplementary Results – Classification of Mutations
Classification of mutations was performed as follows. First, we determined the percentage of
methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) to total cobalamins for each experimental
mutation and control (see above). The percentage MeCbl and AdoCbl for each experimental mutation
was then compared against those from each control: empty vector, wild-type, and cblD-HC, using a twosided T-test (see Table 1). Experimental mutations that had a non-significant difference for both AdoCbl
and MeCbl compared to either vector-only, wild-type or cblD-HC were placed into the category of that
control (e.g. p.D226A showed non-significant differences of MeCbl and AdoCbl levels against cblD-HC
(p.L259P), and was therefore categorized as cblD-HC). If the experimental mutation had a significant
difference for MeCbl and/or AdoCbl levels against all 3 controls, they were then placed into a newly
created category (e.g. p.W189A was significantly different from all 3 controls for both MeCbl and
AdoCbl). Experimental mutations that were significantly different from the controls but were similar to
each other were placed into the same category (e.g. p.P154A, p.E167A and p.K263A all had wild-type like
AdoCbl [non-significant difference] but significantly different MeCbl levels from all 3 controls). Finally,
the three new categories were given names stemming from the cellular or biochemical phenotypes
expected due to the relative levels of MeCbl and AdoCbl, namely: “mild-MMA/HC” for the mutation that
had lower MeCbl and AdoCbl than wild-type vector; “partial cblD-HC” for the mutations that had lower
MeCbl but higher AdoCbl than wild-type (a cblD-HC like pattern, but with MeCbl levels slightly but
significantly different from cblD-HC mutations); and “mild cblD-HC” for those mutations with only slightly
lower MeCbl but much higher AdoCbl levels compared to wild-type (a cblD-HC like pattern, but with
MeCbl severely and significantly different from cblD-HC mutations). The authors note that while the
grouping of these latter three categories was based on statistical comparisons, the naming is arbitrary.
Supplementary Table 1. Primer sequences of generated missense mutations and C-terminal
truncations using site-directed mutagenesis. The sequence of the forward (normal) and reverse
complementary (italic) primers are shown from 5’ to 3’. Base pairs that were changed are marked red.
Codons for mutations are underlined.
Nucleotide change
Protein
c.460C>G
p.P154A
c.493_494delTTinsGC
p.F165A
c.500A>C
p.E167A
c.556_557delATinsGC
p.M186A
c.565_566delTGinsGC
p.W189A
c.589_590delAGinsGC
p.R197A
c.610_611delTTinsGC
p.F204A
c.634_635delTGinsGC
p.C212A
c.677A>C
p.D226A
c.709_710delTAinsGC
p.Y237A
c.787_788deAAlinsGC
p.K263A
c.796_797delCGinsGC
p.R266A
c.808_809delTGinsGC
p.W270A
c.832_833delAGinsGC
p.S278A
c.838_839delTTinsGC
p.F280A
c.783C>A
p.C261*
c.817_819delCATinsTGA
p.H273*
c.826_827delGTinsTG
p.V276*
c.841_843delACTinsTGA
p.T281*
c.859_861delACinsTGA
p.S287*
Primer sequence (5’ to 3’)
GTGTGCAATACAAACATGTGCAGAATTGCTGCGA
ATCTTTTCGCAGCAATTCTGCACATGTTTGTAT
AAAGATTTTGAATCACTGGCTCCAGAAGTAGCT
GCCATTAGCTACTTCTGGAGCCAGTGATTCAAA
TTTGAATCACTGTTTCCAGCAGTAGCTAATGGC
TAGTTTGCCATTAGCTACTGCTGGAAACAGTGA
CAAAAAACTAAGAATGATGCGACTGTTTGGAGTG
CTTCTTCACTCCAAACAGTCGCATCATTCTTAGT
AAGAATGATATGACTGTTGCGAGTGAAGAAGTA
AATTTCTACTTCTTCACTCGCAACAGTCATATC
GAAGAAGTAGAAATTGAAGCAGAAGTGCTCTTA
CTTTTCTAAGAGCACTTCTGCTTCAATTTCTAC
GAAGTGCTCTTAGAAAAGGCCATCAATGGTGCT
TTCCTTAGCACCATTGATGGCCTTTTCTAAGAG
AATGGTGCTAAGGAAATTGCCTATGCTCTTCGA
CTCAGCTCGAAGAGCATAGGCAATTTCCTTAGC
TATTGGGCTGACTTTATTGCCCCATCATCTGGT
TGCCAAACCAGATGATGGGGCAATAAAGTCAG
TTGGCATTTTTTGGACCAGCTACAAACAACACT
AAAAAGAGTGTTGTTTGTAGCTGGTCCAAAAAA
GATGACCTTGGATGCTGTGCAGTGATTCGTCAT
GAGACTATGACGAATCACTGCACAGCATCCAAG
GGATGCTGTAAAGTGATTGCTCATAGTCTCTGG
GGTACCCCAGAGACTATGAGCAATCACTTTACA
GTGATTCGTCATAGTCTCGCGGGTACCCATGTA
TACAACTACATGGGTACCCGCGAGACTATGACG
ACCCATGTAGTTGTAGGGGCTATCTTCACTAAT
TGTTGCATTAGTGAAGATAGCCCCTACAACTAC
GTAGTTGTAGGGAGTATCGCCACTAATGCAACA
GTCTGGTGTTGCATTAGTGGCGATACTCCCTAC
TCTGTTGATGACCTTGGATGATGTAAAGTGATT
ATGACGAATCACTTTACATCATCCAAGGTCATC
ATAGTCTCTGGGGTACCTGAGTAGTTGTAGGG
GATACTCCCTACAACTACTCAGGTACCCCAGAG
TGGGGTACCCATGTAGTTTGAGGGAGTATCTTC
ATTAGTGAAGATACTCCCTCAAACTACATGGGT
TTGTAGGGAGTATCTTCTGAAATGCAACACCA
GCTGTCTGGTGTTGCATTTCAGAAGATACTCCC
ACTAATGCAACACCAGACTGACATATTATGAAG
TAATTTCTTCATAATATGTCAGTCTGGTGTTGC
Location
Exon 5
Exon 5/6
Exon 5/6
Exon 6
Exon 6
Exon 6
Exon 6/7
Exon 7
Exon 7
Exon 7/8
Exon 8
Exon 8
Exon 8
Exon 8
Exon 8
Exon 8
Exon 8
Exon 8
Exon 8
Exon 8