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Accepted Preprint first posted on 21 September 2016 as Manuscript JOE-16-0384
1
A vital region for human glycoprotein hormone trafficking revealed by an LHB mutation
2 1†
2,3,4†
2†
, Michał Kiełbus 4, Krzysztof
3
Iulia Potorac
4
Jozwiak5, Francois Pralong 6, Aicha Hafidi 7, Albert Thiry 8, Jean-Jacques Ménagé 9, Ilpo
5
Huhtaniemi 2,10§, Albert Beckers 1§* and Adrian F. Daly 1§
, Adolfo Rivero-Müller
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
1
25
†
26
equally to the conceptualisation of this work
, Ashutosh Trehan
Department of Endocrinology, Centre Hospitalier Universitaire de Liège, Université de Liège, Domaine Universitaire du Sart-Tilman, 4000 Liège, Belgium 2 Department of Physiology, Institute of Biomedicine, University of Turku, 20520, Turku, Finland 3 Faculty of Natural Sciences and Technology, Åbo Akademi University, 20520, Turku, Finland 4 Department of Biochemistry and Molecular Biology, Medical University of Lublin, 20-093, Lublin, Poland 5 Laboratory of Medicinal Chemistry and Neuroengineering, Medical University of Lublin, Lublin Poland 6 Service of Endocrinology, Diabetology and Metabolism, Department of Medicine, CHU Vaudois, Lausanne, Switzerland 7 Department of Diabetology and Metabolic Diseases, Centre Hospitalier Universitaire Ibn Sina, Rabat, Morocco. 8 Department of Pathology, Centre Hospitalier Universitaire de Liège, Université de Liège, Domaine Universitaire du Sart-Tilman, 4000 Liège, Belgium 9 Endocrinology Practice, Fontainebleau, France 10 Department of Surgery and Cancer, Institute of Reproductive and Developmental Biology, Hammersmith Campus, Imperial College London, London, United Kingdom The first three authors contributed equally to this work; §the last three authors contributed
27 28
Short title: Glycoprotein hormone mutations and secretion
29 30
Word Count: 3150 words
31 32
Keywords: Luteinizing hormone, mutation, hypogonadism, glycoprotein hormone
33 34
*Corresponding author:
35
Professor Albert Beckers, M.D., Ph.D.,
36
Centre Hospitalier Universitaire de Liège,
37
University of Liège,
38
Domaine Universitaire du Sart Tilman,
39
4000 Liege,
40
Belgium
41
Tel: +32 4 366 7083
1 Copyright © 2016 by the Society for Endocrinology.
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Abstract
43
Glycoprotein hormones are complex hormonally active macromolecules. Luteinizing hormone
44
(LH) is essential for the postnatal development and maturation of the male gonad. Inactivating
45
LHB gene mutations are exceptionally rare and lead to hypogonadism that is particularly severe
46
in males. We describe a family with selective LH deficiency and hypogonadism in two brothers.
47
DNA sequencing of LHB was performed and the effects of genetic variants on hormone
48
function and secretion were characterized by mutagenesis studies, confocal microscopy and
49
functional assays. A 20-year-old male from a consanguineous family had pubertal delay,
50
hypogonadism and undetectable LH. A homozygous c.118_120del (p.Lys40del) mutation was
51
identified in the patient and his brother, who subsequently had the same phenotype. Treatment
52
with hCG led to pubertal development, increased circulating testosterone and spermatogenesis.
53
Experiments in HEK293 cells revealed that the mutant LH is retained intracellularly and
54
showed diffuse cytoplasmic distribution. The mutated LHB heterodimerizes with the common
55
alpha subunit and can activate its receptor. Deletion of flanking glutamic acid residues at
56
positions 39 and 41 impair LH to a similar extent as deletion of Lys40. This region is
57
functionally important across all heterodimeric glycoprotein hormones, because deletion of the
58
corresponding residues in hCG, follicle-stimulating hormone and thyroid-stimulating hormone
59
beta-subunits also led to intracellular hormone retention. This novel LHB mutation results in
60
hypogonadism due to intracellular sequestration of the hormone and reveals a discrete region in
61
the protein that is crucial for normal secretion of all human glycoprotein hormones.
62 63
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Introduction
65
Luteinizing hormone (LH) belongs to the heterodimeric glycoprotein hormone family whose
66
other members are human chorionic gonadotropin (hCG), follicle-stimulating hormone (FSH)
67
and thyroid-stimulating hormone (TSH) (Cahoreau et al, 2015). They share a common alpha
68
subunit (CGA) that is non-covalently bound to a hormone-specific beta-subunit. Inactivating
69
germline mutations of beta-subunit genes have been described for all glycoprotein hormones
70
apart from hCG, probably due to the importance of hCG for pregnancy success.
71
Inactivating mutations of the LH beta-subunit gene (LHB) are exceptionally rare and have been
72
reported as homozygous mutations in consanguineous kindreds; a compound heterozygous LHB
73
mutation was also identified (Weiss et al, 1992; Valdes-Socin et al 2004; Lofrano-Porto et al
74
2007; Achard et al 2009; Basciani et al 2012). Affected males have pubertal delay that is
75
usually associated with arrested spermatogenesis and infertility.
76
phenotype with pubertal development and spontaneous menarche that is followed by
77
anovulatory oligomenorrhea/secondary amenorrhea. Males with LHB mutations respond to
78
hCG treatment, that stimulates the LH-hCG receptor (LHCGR) in Leydig cells, leading to
79
increased testosterone production, sexual maturation and improved spermatogenesis (Valdes-
80
Socin et al 2004; Lofrano-Porto et al 2007; Achard et al 2009, Valdes-Socin et al 2009).
81
Study of rare inactivating mutations of glycoprotein hormone beta-subunit genes has provided
82
important insights into their structural-functional relationships, their heterodimerization with
83
CGA, and their receptor interactions. Here, we report a LHB mutation in two homozygous
84
brothers that resulted in a deletion of a lysine at position 40 of the LHB peptide. Unlike other
85
LHB mutations, the mutant LHB was synthesized, formed heterodimers and activated the
86
LHCGR in vitro. The deletion of lysine 40 led to marked intracellular retention of LHB subunit.
87
Similar experimental deletions in the beta subunits of the other glycoprotein hormones (hCG,
88
FSH and TSH) led to an identical pattern of intracellular retention, indicating an important role
89
for this region in intracellular handling and secretion of this important family of hormones.
Females have a milder
90
3
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Methods
92
Mutation detection
93
After obtaining informed consent, genetic and hormonal studies were undertaken in members of
94
the kindred. Genomic DNA was extracted from blood leukocytes and a 1082bp amplicon
95
containing the complete LHB was sequenced. Primer sequences with 3’ end mismatches in the
96
3’ primer (to differentiate LHB from the similar hCGB) are shown in the Supplemental
97
materials. Thirty-one members of the kindred were screened specifically for the mutation found
98
in the propositus and his prepubertal brother (Figure 1C).
99 100
Construction of mutant cDNA expression vectors
101
We first generated the AmCyan-P2A-mCherry vector, where nAmCyan (containing a nuclear
102
localisation signal, NLS) and mCherry are separated by P2A (a 22 amino acid self-cleaving
103
peptide) such that upon expression of this vector, nAmCyan goes to nucleus while mCherry is
104
retained in the cytoplasm (Supplementary Figure S1). The plasmid contains restriction sites to
105
allow insertion of genes of interest to act as fusion proteins with mCherry (with nAmCyan as a
106
reporter), or as splitting proteins from mCherry where nAmCyan is replaced with the gene of
107
interest. The AmCyan-P2A-mCherry plasmid can be accessed via the Addgene repository
108
(Addgene plasmid # 45350; Supplementary Figure S2).
109 110
The LHB wild type (WT)- and mutant (Lys40del)-expressing plasmids were generated by
111
cloning 500 basepair gBlocks (IDT) into the AmCyan-P2A-mCherry as a fusion protein with
112
mCherry via Gibson assembly (NEB). This allows the expression of the LHB gene to be
113
monitored by nuclear-AmCyan, while the LHB-mCherry could be observed during
114
biosynthesis, transport and secretion. The final vectors (AmCyan-P2A-WT LHB-mCherry and
115
AmCyan-P2A-Lys40del LHB-mCherry) were used for all in vitro studies. Similarly, gBlocks of
116
WT FSH beta (FSHB), TSH beta (TSHB) and hCG beta (hCGB) were cloned as fusion proteins
117
with mCherry. In order to determine if the signal peptide of LHB is affected by the downstream
118
amino acids, we replaced the LHB signal peptide in the LHB-Lys40del mutant for that of
4
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prolactin (PRL). This was done by inserting a gBlock coding PRL-LHB-Lys40del into the
120
AmCyan-P2A-mCherry by Gibson assembly as described above. The plasmid carrying CGA
121
under the ubiquitin C promoter has been previously described (Ahtiainen et al 2010).
122 123
Mutagenesis
124
The AmCyan-P2A-WT LHB-mCherry, WT-FSHB, WT-hCGB or WT-TSHB plasmids were
125
used as templates for all further mutations. All mutants were generated by REPLACR
126
technology and were sequence-verified (Trehan et al 2016). Primers for mutagenesis are
127
presented in Supplementary Tables S1 and S2. The sequences of all cloned genes are presented
128
in Supplementary Figure S3, and their gene products in Supplementary Figure S4.
129 130
Signal transduction studies
131
HEK293 cells stably expressing a luminescent cAMP sensor (Glosensor-22F, Promega) and
132
either luteinizing hormone/chorionic gonadotropin receptor (LHCGR) or follicle-stimulating
133
hormone receptor (FSHR) have been previously described 10 and are referred to as GS-LHCGR
134
or GS-FSHR cells, respectively. Similar cAMP sensor cells transiently expressing the thyroid-
135
stimulating hormone receptor (TSHR; GS-TSHR cells) were developed. HEK293 cells were
136
transiently transfected with WT and mutant constructs of LHB, FSHB, TSHB and hCGB, either
137
alone or co-transfected with CGA, and 36h later the cell culture medium was replaced with fresh
138
DMEM/F12 medium (with 5% charcoal-treated serum) for 8h. The medium was then collected
139
and stored at -20 °C and either directly used for cell stimulation or first concentrated using a
140
microconcentrator (Millipore 10 KDa cut-off). Activation of LHCGR, FSHR or TSHR was
141
measured by cAMP production, as a luminescent read-out (relative light units; RLU) and is
142
represented as area under curve. All experiments were independently repeated at least three
143
times in triplicate.
144 145
Confocal microscopy
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AmCyan and mCherry were excited with 458 nm and 543 nm laser lines, and emissions were
147
collected at 470-579 nm and 578-696 nm, respectively. Confocal microscopes (Zeiss LSM780
148
and LSM 510 META) were used for the experiments in living cells. Imaris software was used to
149
export images as 3D volume renders of the Z-stacks. Contrast was increased uniformly on the
150
whole image, for WT LHB, FSHB and hCGB to better represent the secretion vesicles that are
151
heavily secreting their respective hormone subunits out of the cells, thus retaining very small
152
quantities of mCherry-tagged hormones inside the cells.
153 154
Measurement of LHB and LHB/CGA
155
LHB and LHB/CGA were measured using commercial ELISA kits: DELFIA hLH Spec that
156
used an immobilized antibody against hLHB and a labelled secondary antibody against a
157
different hLHB or DELFIA hLH SPEC immobilized primary antibody with at labelled
158
secondary antibody of DELFIA hFSH which detects the common CGA, respectively)
159
(PerkinElmer).
160 161
Three-dimentional (3D) modelling
162
Since no crystal structure for the target, LHB, is avalable, we used X-ray crystallography
163
structure of homologous hCG beta (1HRP.pdb) as a template. The residues which are not
164
evolutionary conserved between the target and the template were replaced. All modeling and
165
visualization was done using YASARA 11.11.2. package.
166 167
Diffusion assay: Fluorescence Recovery After Photobleaching (FRAP)
168
Each FRAP experiment started with an image acquisition of a cell’s cytoplasm using 561 nm
169
laser (525/50 nm excitation and 595/50 nm emission filter blocks) followed by a single-point
170
bleach (nonscanning) in the cytoplasm area at full power of 405 nm laser for the minimum time
171
required to photobleach most of cytoplasmic fluorescence (1-5 s). Fluorescence recovery was
172
measured by starting a time-lapse acquisition within 0.25 s after bleaching. Image size was 512
173
× 512 pixels and 450 scans over 120 s were performed. Pinhole size was set to the optimal value
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(12.77 µm confocal aperture). Normalized fluorescence was calculated as the ratio of intensity
175
of bleached region fluorescence to intensity of reference region fluorescence. The lowest value
176
of fluorescence intensity was estimated as 0.
177 178
Statistical Analysis
179
Statistical analyses were performed using GraphPad Prism 6 software using one-way ANOVA.
180
Statistical significance and p-values are indicated in the respective figures.
181
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182
Results
183
Clinical studies
184
A 20-year-old male of North-African descent was referred for investigation of pubertal delay.
185
He presented with a hypogonadal phenotype: tall stature (190cm), scant androgen-dependent
186
pilosity, bilateral gynecomastia, micropenis and low testicular volume (~7ml) (Figure 1A). He
187
reported low libido and infrequent erections. Laboratory tests confirmed hypogonadism [total
188
testosterone 0.64 ng/mL, reference range (RR): 2.5-10; 2.2 nmol/L, RR: 8.7-37.4] with low LH
189
(0.4 IU/L, RR: 2-10), high FSH (19.6 IU/L, RR:1-8) and high free α-subunit (3.6 IU/L, RR
190
males 230-fold
280
less than WT-LHB, leading to decreased circulating LH that caused hypogonadism in the
281
affected patients.
282
activating the LHCGR.
283
Male sexual differentiation, development and reproductive function are strictly related to
284
androgen secretion and action. Secretion of androgens depends on the functionality of the hCG-
285
LH-LHCGR axis. In humans, placental hCG binds to the LHCGR, stimulating fetal Leydig cells
286
to secrete testosterone during fetal development, thus ensuring stabilization of the Wolffian
287
ducts and their differentiation into the internal male genital tract (Themmen et al 2000).
288
Following the conversion of testosterone to dihydrotestosterone the latter induces the
289
development of the prostate and external genital organs.
290
LHCGR mutations have more severe phenotype at birth (e.g. pseudohermaphroditism) than
291
those with inactivating LHB mutations that are born normal (Latronico and Arnhold 2013).
292
Post-natally, LH becomes the LHCGR agonist, exerting its effect on adult Leydig cells from
293
puberty onwards. To date, only five other inactivating mutations of the LHB have been
294
described (Weiss et al, 1992; Valdes-Socin et al 2004; Lofrano-Porto et al 2007; Achard et al
295
2009; Basciani et al 2012). Their clinical presentation is similar with pubertal arrest in males
296
(Axelrod et al, 1979; Valdes-Socin et al 2004; Lofrano-Porto et al 2007; Achard et al 2009;
297
Basciani et al 2012) and secondary amenorrhea/oligomenorrhea, anovulation and infertility in
298
females despite normal secondary sexual characteristics (Lofrano-Porto et al 2007; Achard et al
299
2009; Basciani et al 2012). Due to the more dramatic clinical presentation, LHB mutations were
The mutated LHB is, however, capable of dimerizing with CGA and
Hence, males with inactivating
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300
initially found only in males; the female cases described are sisters of male index patients.
301
Treatment of the two patients described here with hCG normalized testosterone secretion,
302
thereby enabling the acquisition of secondary sexual characteristics and improvement in sperm
303
production.
304
Previous instances of LH inactivation were due to homozygous mutations that prevented
305
binding to LHCGR (Axelrod et al 1979; Weiss et al 1992) or the formation of heterodimers
306
(Valdes-Socin et al, 2004), deleted important cysteine residues (H30-I32del) (Achard et al
307
2009), caused abnormal splicing of the LH beta subunit (Lofrano-Porto et al 2007), or presented
308
with a compound heterozygous mutation of the signal peptide (L9-L12del) in combination with
309
a mutation at an intronic splice donor site on the other allele (Basciani et al 2012).
310
The genetic mechanism impairing LH action in this family differs from the others described
311
previously. We showed that the Lys40del did not impair the expression of LHB, prevent
312
heterodimerization or alter the ratio of LHB monomer to dimer. Furthermore, the mutated
313
dimer bound to and activated its receptor, LHCGR. Faulty trafficking of the mutated dimeric
314
LH caused absent LH secretion; instead of targeted transport to secretory vesicles, the mutated
315
LH diffused generally into the cytoplasm. Substituting Lys40 with either a hydrophilic
316
(arginine), a hydrophobic (alanine) or a neutral residue (asparagine) did not impair LH function.
317
Deleting either of the glutamic acids that flank Lys40 resulted in an identical effect on receptor
318
stimulation to that seen with Lys40del. This indicates that amino acids in this region are crucial
319
for correct intracellular trafficking of LH during its synthesis, and deletions result in
320
intracellular retention, lack of LH secretion and the observed clinical phenotype. The 3D model
321
indicates that Lys40 is situated in a loop between alpha helices and alterations in this loop
322
appear to trigger a change in the peptide 3D structure.
323
Although the glycoprotein hormone family regulates diverse biological functions, the individual
324
hormones share a significant structural similarity (Li and Ford 1998). The CGA and beta
325
subunits belong to the cystine-knot superfamily and have a similar tertiary structure with three
326
elongated loops (Cahoreau et al 2015). The C-terminal segment of the beta-subunit stabilizes
327
the heterodimer by wrapping around the alpha-subunit like a seat-belt and latching through a
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disulphide bond with a cysteine residue from loop 1 of the beta-subunit (Lapthorn et al 1994;
329
Xing et al 2001; Xing et al 2004). Lys40 is situated in the beta-1 loop, close to a cysteine
330
residue that is part of the seat-belt latch. However, deletion of Lys40 or its flanking glutamic
331
acid residues appears not to impair the assembly of functional dimeric LH.
332
analogous mutations of hCG, FSH and TSH beta subunits had a very similar effect to that seen
333
in LH, with marked cytoplasmic retention, minimal secretion and decreased receptor
334
stimulation. This general effect of deletions in this discrete region across the glycoprotein beta-
335
subunits suggests impairment of a common step that precedes trafficking of hormones to
336
secretory vesicles. This is most likely due to protein misfolding after the protein is undocked
337
from the endoplasmic reticulum and before it enters the Golgi apparatus.
338
diffusion of mutated protein seen on confocal microscopy for all the glycoprotein hormones
339
suggests that misfolded protein has been re-exported out of the endoplasmic reticulum. While
340
we would speculate that the misfolded glycoprotein hormone would be undergo ubiquitination
341
and proteasomal destruction, their continued presence throughout the cytoplasm argues against
342
rapid degradation (Smoth et al 2011).
343
In conclusion, the clinical phenotype of hypogonadism in two brothers with a homozygous LHB
344
mutation causing a single amino acid deletion reveals an important region for normal secretion
345
of all four human glycoprotein hormones. These discrete changes led to intracellular retention
346
of functional dimeric hormones that can have important clinical consequences. Additionally, we
347
have developed a new system to test mutations in glycoprotein hormones that allows for easy
348
visualization of expression and localization.
Intriguingly,
The cytosolic
349
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Acknowledgements
413
The authors would like to acknowledge Dr. Roberto Salvi and Dr. Maria Cristina Burlacu for
414
contributions to the genetic and clinical studies. The study was supported by grants from the
415
Academy of Finland, Turku Doctoral Programme of Biomedical Sciences (TuBS), Turku
416
Doctoral Programme of Molecular Medicine (TuDMM), the Sigrid Jusélius Foundation, the
417
Polish National Science Center UMO-2015/17/B/NZ1/01777, and the Fonds d’Investissement
418
pour la Recherche (FIRS) of the Centre Hospitalier Universitaire de Liège, Belgium.
419 420
Author Contributions
421
A.F.D., A.R-M. and I.P. designed the experiments. I.P., A.H., J-J.M., F.P. A.B., and A.F.D.,
422
designed, performed or oversaw clinical and genetic studies. A.R-M. and A.T. participated in
423
design and construction of plasmid vectors and conducted the functional tests. A.T. participated
424
in visualization of beta subunits by confocal microscopy. A.Th. conducted pathological studies
425
of testicular biopsy. M.K. generated the diffusion data. K.J. performed the 3D modelling. I.P.,
426
A.R-M. A.T. and A.F.D. wrote the manuscript. I.H. and A.B. coordinated the research. All
427
authors read and approved the final manuscript.
428 429
Competing Financial Interests
430
The authors report that they have no relevant conflicts of interest.
431 432
Materials and Correspondence
433
Any request for materials used in the manuscript can be forwarded to A.T., A.R-M., A.F.D or
434
A.B.
435 436
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Page 18 of 28
Legends
Figure 1. Clinical characterization A and B) External genitalia of the patient before and after treatment with hCG for 12 months is shown, with evident increase in penile length, testicular volume and pilosity. C) Kindred diagram showing consanguinity; carriers and affected patients with the p.Lys40del mutation of LHB are indicated. D) Testicular biopsy (hematoxylin & eosin staining, 100x) showing seminiferous tubules with globally thickened basement membrane (black arrow). Spermatogenesis is present but arrested at the round spermatid stage (white arrow) and few Leydig cells are seen in the interstitial space (open arrow).
Figure 2. Functional and visual characterization of the LHB-Lys40del as compared to WTLHB. A) Medium was collected from HEK293 cells transfected with various plasmid combinations and used to stimulate LHCGR using GS-LHCGR reporter cells. The luminescent read-out is proportional to cAMP production. CGA alone, LHB alone or medium from untransfected cells (medium alone) resulted in no activation of the LHCGR. WT-LHB+CGA produced the expected activation, while the LHB-Lys40del+CGA mutant showed a minimal, significantly reduced response (p-value
Accepted Preprint first posted on 21 September 2016 as Manuscript JOE-16-0384
1
A vital region for human glycoprotein hormone trafficking revealed by an LHB mutation
2 1†
2,3,4†
2†
, Michał Kiełbus 4, Krzysztof
3
Iulia Potorac
4
Jozwiak5, Francois Pralong 6, Aicha Hafidi 7, Albert Thiry 8, Jean-Jacques Ménagé 9, Ilpo
5
Huhtaniemi 2,10§, Albert Beckers 1§* and Adrian F. Daly 1§
, Adolfo Rivero-Müller
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
1
25
†
26
equally to the conceptualisation of this work
, Ashutosh Trehan
Department of Endocrinology, Centre Hospitalier Universitaire de Liège, Université de Liège, Domaine Universitaire du Sart-Tilman, 4000 Liège, Belgium 2 Department of Physiology, Institute of Biomedicine, University of Turku, 20520, Turku, Finland 3 Faculty of Natural Sciences and Technology, Åbo Akademi University, 20520, Turku, Finland 4 Department of Biochemistry and Molecular Biology, Medical University of Lublin, 20-093, Lublin, Poland 5 Laboratory of Medicinal Chemistry and Neuroengineering, Medical University of Lublin, Lublin Poland 6 Service of Endocrinology, Diabetology and Metabolism, Department of Medicine, CHU Vaudois, Lausanne, Switzerland 7 Department of Diabetology and Metabolic Diseases, Centre Hospitalier Universitaire Ibn Sina, Rabat, Morocco. 8 Department of Pathology, Centre Hospitalier Universitaire de Liège, Université de Liège, Domaine Universitaire du Sart-Tilman, 4000 Liège, Belgium 9 Endocrinology Practice, Fontainebleau, France 10 Department of Surgery and Cancer, Institute of Reproductive and Developmental Biology, Hammersmith Campus, Imperial College London, London, United Kingdom The first three authors contributed equally to this work; §the last three authors contributed
27 28
Short title: Glycoprotein hormone mutations and secretion
29 30
Word Count: 3150 words
31 32
Keywords: Luteinizing hormone, mutation, hypogonadism, glycoprotein hormone
33 34
*Corresponding author:
35
Professor Albert Beckers, M.D., Ph.D.,
36
Centre Hospitalier Universitaire de Liège,
37
University of Liège,
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Domaine Universitaire du Sart Tilman,
39
4000 Liege,
40
Belgium
41
Tel: +32 4 366 7083
1 Copyright © 2016 by the Society for Endocrinology.
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Abstract
43
Glycoprotein hormones are complex hormonally active macromolecules. Luteinizing hormone
44
(LH) is essential for the postnatal development and maturation of the male gonad. Inactivating
45
LHB gene mutations are exceptionally rare and lead to hypogonadism that is particularly severe
46
in males. We describe a family with selective LH deficiency and hypogonadism in two brothers.
47
DNA sequencing of LHB was performed and the effects of genetic variants on hormone
48
function and secretion were characterized by mutagenesis studies, confocal microscopy and
49
functional assays. A 20-year-old male from a consanguineous family had pubertal delay,
50
hypogonadism and undetectable LH. A homozygous c.118_120del (p.Lys40del) mutation was
51
identified in the patient and his brother, who subsequently had the same phenotype. Treatment
52
with hCG led to pubertal development, increased circulating testosterone and spermatogenesis.
53
Experiments in HEK293 cells revealed that the mutant LH is retained intracellularly and
54
showed diffuse cytoplasmic distribution. The mutated LHB heterodimerizes with the common
55
alpha subunit and can activate its receptor. Deletion of flanking glutamic acid residues at
56
positions 39 and 41 impair LH to a similar extent as deletion of Lys40. This region is
57
functionally important across all heterodimeric glycoprotein hormones, because deletion of the
58
corresponding residues in hCG, follicle-stimulating hormone and thyroid-stimulating hormone
59
beta-subunits also led to intracellular hormone retention. This novel LHB mutation results in
60
hypogonadism due to intracellular sequestration of the hormone and reveals a discrete region in
61
the protein that is crucial for normal secretion of all human glycoprotein hormones.
62 63
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Introduction
65
Luteinizing hormone (LH) belongs to the heterodimeric glycoprotein hormone family whose
66
other members are human chorionic gonadotropin (hCG), follicle-stimulating hormone (FSH)
67
and thyroid-stimulating hormone (TSH) (Cahoreau et al, 2015). They share a common alpha
68
subunit (CGA) that is non-covalently bound to a hormone-specific beta-subunit. Inactivating
69
germline mutations of beta-subunit genes have been described for all glycoprotein hormones
70
apart from hCG, probably due to the importance of hCG for pregnancy success.
71
Inactivating mutations of the LH beta-subunit gene (LHB) are exceptionally rare and have been
72
reported as homozygous mutations in consanguineous kindreds; a compound heterozygous LHB
73
mutation was also identified (Weiss et al, 1992; Valdes-Socin et al 2004; Lofrano-Porto et al
74
2007; Achard et al 2009; Basciani et al 2012). Affected males have pubertal delay that is
75
usually associated with arrested spermatogenesis and infertility.
76
phenotype with pubertal development and spontaneous menarche that is followed by
77
anovulatory oligomenorrhea/secondary amenorrhea. Males with LHB mutations respond to
78
hCG treatment, that stimulates the LH-hCG receptor (LHCGR) in Leydig cells, leading to
79
increased testosterone production, sexual maturation and improved spermatogenesis (Valdes-
80
Socin et al 2004; Lofrano-Porto et al 2007; Achard et al 2009, Valdes-Socin et al 2009).
81
Study of rare inactivating mutations of glycoprotein hormone beta-subunit genes has provided
82
important insights into their structural-functional relationships, their heterodimerization with
83
CGA, and their receptor interactions. Here, we report a LHB mutation in two homozygous
84
brothers that resulted in a deletion of a lysine at position 40 of the LHB peptide. Unlike other
85
LHB mutations, the mutant LHB was synthesized, formed heterodimers and activated the
86
LHCGR in vitro. The deletion of lysine 40 led to marked intracellular retention of LHB subunit.
87
Similar experimental deletions in the beta subunits of the other glycoprotein hormones (hCG,
88
FSH and TSH) led to an identical pattern of intracellular retention, indicating an important role
89
for this region in intracellular handling and secretion of this important family of hormones.
Females have a milder
90
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Methods
92
Mutation detection
93
After obtaining informed consent, genetic and hormonal studies were undertaken in members of
94
the kindred. Genomic DNA was extracted from blood leukocytes and a 1082bp amplicon
95
containing the complete LHB was sequenced. Primer sequences with 3’ end mismatches in the
96
3’ primer (to differentiate LHB from the similar hCGB) are shown in the Supplemental
97
materials. Thirty-one members of the kindred were screened specifically for the mutation found
98
in the propositus and his prepubertal brother (Figure 1C).
99 100
Construction of mutant cDNA expression vectors
101
We first generated the AmCyan-P2A-mCherry vector, where nAmCyan (containing a nuclear
102
localisation signal, NLS) and mCherry are separated by P2A (a 22 amino acid self-cleaving
103
peptide) such that upon expression of this vector, nAmCyan goes to nucleus while mCherry is
104
retained in the cytoplasm (Supplementary Figure S1). The plasmid contains restriction sites to
105
allow insertion of genes of interest to act as fusion proteins with mCherry (with nAmCyan as a
106
reporter), or as splitting proteins from mCherry where nAmCyan is replaced with the gene of
107
interest. The AmCyan-P2A-mCherry plasmid can be accessed via the Addgene repository
108
(Addgene plasmid # 45350; Supplementary Figure S2).
109 110
The LHB wild type (WT)- and mutant (Lys40del)-expressing plasmids were generated by
111
cloning 500 basepair gBlocks (IDT) into the AmCyan-P2A-mCherry as a fusion protein with
112
mCherry via Gibson assembly (NEB). This allows the expression of the LHB gene to be
113
monitored by nuclear-AmCyan, while the LHB-mCherry could be observed during
114
biosynthesis, transport and secretion. The final vectors (AmCyan-P2A-WT LHB-mCherry and
115
AmCyan-P2A-Lys40del LHB-mCherry) were used for all in vitro studies. Similarly, gBlocks of
116
WT FSH beta (FSHB), TSH beta (TSHB) and hCG beta (hCGB) were cloned as fusion proteins
117
with mCherry. In order to determine if the signal peptide of LHB is affected by the downstream
118
amino acids, we replaced the LHB signal peptide in the LHB-Lys40del mutant for that of
4
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prolactin (PRL). This was done by inserting a gBlock coding PRL-LHB-Lys40del into the
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AmCyan-P2A-mCherry by Gibson assembly as described above. The plasmid carrying CGA
121
under the ubiquitin C promoter has been previously described (Ahtiainen et al 2010).
122 123
Mutagenesis
124
The AmCyan-P2A-WT LHB-mCherry, WT-FSHB, WT-hCGB or WT-TSHB plasmids were
125
used as templates for all further mutations. All mutants were generated by REPLACR
126
technology and were sequence-verified (Trehan et al 2016). Primers for mutagenesis are
127
presented in Supplementary Tables S1 and S2. The sequences of all cloned genes are presented
128
in Supplementary Figure S3, and their gene products in Supplementary Figure S4.
129 130
Signal transduction studies
131
HEK293 cells stably expressing a luminescent cAMP sensor (Glosensor-22F, Promega) and
132
either luteinizing hormone/chorionic gonadotropin receptor (LHCGR) or follicle-stimulating
133
hormone receptor (FSHR) have been previously described 10 and are referred to as GS-LHCGR
134
or GS-FSHR cells, respectively. Similar cAMP sensor cells transiently expressing the thyroid-
135
stimulating hormone receptor (TSHR; GS-TSHR cells) were developed. HEK293 cells were
136
transiently transfected with WT and mutant constructs of LHB, FSHB, TSHB and hCGB, either
137
alone or co-transfected with CGA, and 36h later the cell culture medium was replaced with fresh
138
DMEM/F12 medium (with 5% charcoal-treated serum) for 8h. The medium was then collected
139
and stored at -20 °C and either directly used for cell stimulation or first concentrated using a
140
microconcentrator (Millipore 10 KDa cut-off). Activation of LHCGR, FSHR or TSHR was
141
measured by cAMP production, as a luminescent read-out (relative light units; RLU) and is
142
represented as area under curve. All experiments were independently repeated at least three
143
times in triplicate.
144 145
Confocal microscopy
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AmCyan and mCherry were excited with 458 nm and 543 nm laser lines, and emissions were
147
collected at 470-579 nm and 578-696 nm, respectively. Confocal microscopes (Zeiss LSM780
148
and LSM 510 META) were used for the experiments in living cells. Imaris software was used to
149
export images as 3D volume renders of the Z-stacks. Contrast was increased uniformly on the
150
whole image, for WT LHB, FSHB and hCGB to better represent the secretion vesicles that are
151
heavily secreting their respective hormone subunits out of the cells, thus retaining very small
152
quantities of mCherry-tagged hormones inside the cells.
153 154
Measurement of LHB and LHB/CGA
155
LHB and LHB/CGA were measured using commercial ELISA kits: DELFIA hLH Spec that
156
used an immobilized antibody against hLHB and a labelled secondary antibody against a
157
different hLHB or DELFIA hLH SPEC immobilized primary antibody with at labelled
158
secondary antibody of DELFIA hFSH which detects the common CGA, respectively)
159
(PerkinElmer).
160 161
Three-dimentional (3D) modelling
162
Since no crystal structure for the target, LHB, is avalable, we used X-ray crystallography
163
structure of homologous hCG beta (1HRP.pdb) as a template. The residues which are not
164
evolutionary conserved between the target and the template were replaced. All modeling and
165
visualization was done using YASARA 11.11.2. package.
166 167
Diffusion assay: Fluorescence Recovery After Photobleaching (FRAP)
168
Each FRAP experiment started with an image acquisition of a cell’s cytoplasm using 561 nm
169
laser (525/50 nm excitation and 595/50 nm emission filter blocks) followed by a single-point
170
bleach (nonscanning) in the cytoplasm area at full power of 405 nm laser for the minimum time
171
required to photobleach most of cytoplasmic fluorescence (1-5 s). Fluorescence recovery was
172
measured by starting a time-lapse acquisition within 0.25 s after bleaching. Image size was 512
173
× 512 pixels and 450 scans over 120 s were performed. Pinhole size was set to the optimal value
6
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174
(12.77 µm confocal aperture). Normalized fluorescence was calculated as the ratio of intensity
175
of bleached region fluorescence to intensity of reference region fluorescence. The lowest value
176
of fluorescence intensity was estimated as 0.
177 178
Statistical Analysis
179
Statistical analyses were performed using GraphPad Prism 6 software using one-way ANOVA.
180
Statistical significance and p-values are indicated in the respective figures.
181
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182
Results
183
Clinical studies
184
A 20-year-old male of North-African descent was referred for investigation of pubertal delay.
185
He presented with a hypogonadal phenotype: tall stature (190cm), scant androgen-dependent
186
pilosity, bilateral gynecomastia, micropenis and low testicular volume (~7ml) (Figure 1A). He
187
reported low libido and infrequent erections. Laboratory tests confirmed hypogonadism [total
188
testosterone 0.64 ng/mL, reference range (RR): 2.5-10; 2.2 nmol/L, RR: 8.7-37.4] with low LH
189
(0.4 IU/L, RR: 2-10), high FSH (19.6 IU/L, RR:1-8) and high free α-subunit (3.6 IU/L, RR
190
males 230-fold
280
less than WT-LHB, leading to decreased circulating LH that caused hypogonadism in the
281
affected patients.
282
activating the LHCGR.
283
Male sexual differentiation, development and reproductive function are strictly related to
284
androgen secretion and action. Secretion of androgens depends on the functionality of the hCG-
285
LH-LHCGR axis. In humans, placental hCG binds to the LHCGR, stimulating fetal Leydig cells
286
to secrete testosterone during fetal development, thus ensuring stabilization of the Wolffian
287
ducts and their differentiation into the internal male genital tract (Themmen et al 2000).
288
Following the conversion of testosterone to dihydrotestosterone the latter induces the
289
development of the prostate and external genital organs.
290
LHCGR mutations have more severe phenotype at birth (e.g. pseudohermaphroditism) than
291
those with inactivating LHB mutations that are born normal (Latronico and Arnhold 2013).
292
Post-natally, LH becomes the LHCGR agonist, exerting its effect on adult Leydig cells from
293
puberty onwards. To date, only five other inactivating mutations of the LHB have been
294
described (Weiss et al, 1992; Valdes-Socin et al 2004; Lofrano-Porto et al 2007; Achard et al
295
2009; Basciani et al 2012). Their clinical presentation is similar with pubertal arrest in males
296
(Axelrod et al, 1979; Valdes-Socin et al 2004; Lofrano-Porto et al 2007; Achard et al 2009;
297
Basciani et al 2012) and secondary amenorrhea/oligomenorrhea, anovulation and infertility in
298
females despite normal secondary sexual characteristics (Lofrano-Porto et al 2007; Achard et al
299
2009; Basciani et al 2012). Due to the more dramatic clinical presentation, LHB mutations were
The mutated LHB is, however, capable of dimerizing with CGA and
Hence, males with inactivating
12
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300
initially found only in males; the female cases described are sisters of male index patients.
301
Treatment of the two patients described here with hCG normalized testosterone secretion,
302
thereby enabling the acquisition of secondary sexual characteristics and improvement in sperm
303
production.
304
Previous instances of LH inactivation were due to homozygous mutations that prevented
305
binding to LHCGR (Axelrod et al 1979; Weiss et al 1992) or the formation of heterodimers
306
(Valdes-Socin et al, 2004), deleted important cysteine residues (H30-I32del) (Achard et al
307
2009), caused abnormal splicing of the LH beta subunit (Lofrano-Porto et al 2007), or presented
308
with a compound heterozygous mutation of the signal peptide (L9-L12del) in combination with
309
a mutation at an intronic splice donor site on the other allele (Basciani et al 2012).
310
The genetic mechanism impairing LH action in this family differs from the others described
311
previously. We showed that the Lys40del did not impair the expression of LHB, prevent
312
heterodimerization or alter the ratio of LHB monomer to dimer. Furthermore, the mutated
313
dimer bound to and activated its receptor, LHCGR. Faulty trafficking of the mutated dimeric
314
LH caused absent LH secretion; instead of targeted transport to secretory vesicles, the mutated
315
LH diffused generally into the cytoplasm. Substituting Lys40 with either a hydrophilic
316
(arginine), a hydrophobic (alanine) or a neutral residue (asparagine) did not impair LH function.
317
Deleting either of the glutamic acids that flank Lys40 resulted in an identical effect on receptor
318
stimulation to that seen with Lys40del. This indicates that amino acids in this region are crucial
319
for correct intracellular trafficking of LH during its synthesis, and deletions result in
320
intracellular retention, lack of LH secretion and the observed clinical phenotype. The 3D model
321
indicates that Lys40 is situated in a loop between alpha helices and alterations in this loop
322
appear to trigger a change in the peptide 3D structure.
323
Although the glycoprotein hormone family regulates diverse biological functions, the individual
324
hormones share a significant structural similarity (Li and Ford 1998). The CGA and beta
325
subunits belong to the cystine-knot superfamily and have a similar tertiary structure with three
326
elongated loops (Cahoreau et al 2015). The C-terminal segment of the beta-subunit stabilizes
327
the heterodimer by wrapping around the alpha-subunit like a seat-belt and latching through a
13
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328
disulphide bond with a cysteine residue from loop 1 of the beta-subunit (Lapthorn et al 1994;
329
Xing et al 2001; Xing et al 2004). Lys40 is situated in the beta-1 loop, close to a cysteine
330
residue that is part of the seat-belt latch. However, deletion of Lys40 or its flanking glutamic
331
acid residues appears not to impair the assembly of functional dimeric LH.
332
analogous mutations of hCG, FSH and TSH beta subunits had a very similar effect to that seen
333
in LH, with marked cytoplasmic retention, minimal secretion and decreased receptor
334
stimulation. This general effect of deletions in this discrete region across the glycoprotein beta-
335
subunits suggests impairment of a common step that precedes trafficking of hormones to
336
secretory vesicles. This is most likely due to protein misfolding after the protein is undocked
337
from the endoplasmic reticulum and before it enters the Golgi apparatus.
338
diffusion of mutated protein seen on confocal microscopy for all the glycoprotein hormones
339
suggests that misfolded protein has been re-exported out of the endoplasmic reticulum. While
340
we would speculate that the misfolded glycoprotein hormone would be undergo ubiquitination
341
and proteasomal destruction, their continued presence throughout the cytoplasm argues against
342
rapid degradation (Smoth et al 2011).
343
In conclusion, the clinical phenotype of hypogonadism in two brothers with a homozygous LHB
344
mutation causing a single amino acid deletion reveals an important region for normal secretion
345
of all four human glycoprotein hormones. These discrete changes led to intracellular retention
346
of functional dimeric hormones that can have important clinical consequences. Additionally, we
347
have developed a new system to test mutations in glycoprotein hormones that allows for easy
348
visualization of expression and localization.
Intriguingly,
The cytosolic
349
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350
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Acknowledgements
413
The authors would like to acknowledge Dr. Roberto Salvi and Dr. Maria Cristina Burlacu for
414
contributions to the genetic and clinical studies. The study was supported by grants from the
415
Academy of Finland, Turku Doctoral Programme of Biomedical Sciences (TuBS), Turku
416
Doctoral Programme of Molecular Medicine (TuDMM), the Sigrid Jusélius Foundation, the
417
Polish National Science Center UMO-2015/17/B/NZ1/01777, and the Fonds d’Investissement
418
pour la Recherche (FIRS) of the Centre Hospitalier Universitaire de Liège, Belgium.
419 420
Author Contributions
421
A.F.D., A.R-M. and I.P. designed the experiments. I.P., A.H., J-J.M., F.P. A.B., and A.F.D.,
422
designed, performed or oversaw clinical and genetic studies. A.R-M. and A.T. participated in
423
design and construction of plasmid vectors and conducted the functional tests. A.T. participated
424
in visualization of beta subunits by confocal microscopy. A.Th. conducted pathological studies
425
of testicular biopsy. M.K. generated the diffusion data. K.J. performed the 3D modelling. I.P.,
426
A.R-M. A.T. and A.F.D. wrote the manuscript. I.H. and A.B. coordinated the research. All
427
authors read and approved the final manuscript.
428 429
Competing Financial Interests
430
The authors report that they have no relevant conflicts of interest.
431 432
Materials and Correspondence
433
Any request for materials used in the manuscript can be forwarded to A.T., A.R-M., A.F.D or
434
A.B.
435 436
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Page 18 of 28
Legends
Figure 1. Clinical characterization A and B) External genitalia of the patient before and after treatment with hCG for 12 months is shown, with evident increase in penile length, testicular volume and pilosity. C) Kindred diagram showing consanguinity; carriers and affected patients with the p.Lys40del mutation of LHB are indicated. D) Testicular biopsy (hematoxylin & eosin staining, 100x) showing seminiferous tubules with globally thickened basement membrane (black arrow). Spermatogenesis is present but arrested at the round spermatid stage (white arrow) and few Leydig cells are seen in the interstitial space (open arrow).
Figure 2. Functional and visual characterization of the LHB-Lys40del as compared to WTLHB. A) Medium was collected from HEK293 cells transfected with various plasmid combinations and used to stimulate LHCGR using GS-LHCGR reporter cells. The luminescent read-out is proportional to cAMP production. CGA alone, LHB alone or medium from untransfected cells (medium alone) resulted in no activation of the LHCGR. WT-LHB+CGA produced the expected activation, while the LHB-Lys40del+CGA mutant showed a minimal, significantly reduced response (p-value
