基于硒代胱氨酸荧光检测与结构预测的胱氨酸尿症相关转运体变异功能分析

何小柏; 钱欣怡; 郑晓光; 张弘; 邵锦邦; 陈小攀; 阮琦; 吕建新; 杨雷香; 陈林洁

doi:10.1631/jzus.B2500767

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基于硒代胱氨酸荧光检测与结构预测的胱氨酸尿症相关转运体变异功能分析

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Research Article | Updated：2026-07-29

基于硒代胱氨酸荧光检测与结构预测的胱氨酸尿症相关转运体变异功能分析
Selenocystine-based fluorescence assay combined with structural prediction for functional analysis of cystine transporter variants in cystinuria
浙江大学学报（英文版）（B辑：生物医学和生物技术） 2026年27卷第7期页码：806-816
Affiliations：

1.School of Laboratory Medicine and Bioengineering, Zhejiang Provincial People’s Hospital, Hangzhou Medical College, Hangzhou 311399, China
2.Department of Genetic and Genomic Medicine, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College, Hangzhou 310014, China
3.Zhejiang Provincial Engineering Research Centre for Key Technology of Diagnostic Testing, Hangzhou 310053, China
Funds：

the Zhejiang Provincial Natural Science Foundation of China(LMS25H160004);the Zhejiang Provincial Medical and Health Science and Technology Programs(2023KY650;2021KY080)
DOI：10.1631/jzus.B2500767
中图分类号：
CSTR：
收稿：2025-11-25，

修回：2026-05-13，

网络首发：2026-07-29，

纸质出版：2026-07-15
Accepted：

何小柏,钱欣怡,郑晓光等.基于硒代胱氨酸荧光检测与结构预测的胱氨酸尿症相关转运体变异功能分析[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2026,27(07):806-816.

Xiaobai HE, Xinyi QIAN, Xiaoguang ZHENG, et al. Selenocystine-based fluorescence assay combined with structural prediction for functional analysis of cystine transporter variants in cystinuria[J]. Journal of Zhejiang University-SCIENCE B, 2026, 27(7): 806-816.
何小柏,钱欣怡,郑晓光等.基于硒代胱氨酸荧光检测与结构预测的胱氨酸尿症相关转运体变异功能分析[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2026,27(07):806-816. DOI： 10.1631/jzus.B2500767.

Xiaobai HE, Xinyi QIAN, Xiaoguang ZHENG, et al. Selenocystine-based fluorescence assay combined with structural prediction for functional analysis of cystine transporter variants in cystinuria[J]. Journal of Zhejiang University-SCIENCE B, 2026, 27(7): 806-816. DOI： 10.1631/jzus.B2500767.

摘要

目的

胱氨酸结石约占成人肾结石的1%–2%，在儿童中可达10%，其病因为胱氨酸尿症——一种由

SLC3A1

和

SLC7A9

基因突变所致的常染色体隐性遗传病。上述突变导致

胱氨酸重吸收障碍，尿中胱氨酸浓度升高，进而促进结石形成。目前临床检测手段难以精确评估不同突变对转运蛋白功能的影响。为此，本研究旨在建立一种无放射性、基于细胞的功能评估体系，以实现对胱氨酸转运蛋白活性及突变致病变异的系统鉴定。

方法

将野生型或突变型

SLC3A1

与

SLC7A9

质粒共转染至HEK293细胞，瞬时表达胱氨酸转运复合体。方法学上整合两条技术路径：其一，基于硒代胱氨酸的荧光摄取实验，利用其作为胱氨酸的功能性类似物，借助固有荧光特性实现转运活性的实时定量检测，规避传统放射性底物法的操作繁琐与辐射安全问题；其二，联合AlphaFold3进行蛋白质结构预测及分子对接，从分子层面解析突变导致功能丧失的机制。

结果

SLC3A1/SLC7A9复合体对硒代胱氨酸的表观米氏常数（

=(156.3±24.2) μmol/L)与文献报道的天然底物胱氨酸（约200 μmol/L）高度接近，验证了替代底物的可行性。依据残余活性阈值（轻度

60%；中度20%–60%；重度

20%），对8种临床相关变异体（

SLC7A9

：A70V、A182T、G105R、R333W、V170M、A354T、P482L；

SLC3A1

：M467T）进行功能分级，结果与既往放射性同位素法数据一致。AlphaFold3建模显示，P482L可能破坏跨膜区螺旋堆积，A354T则可能干扰氢键网络，为功能丧失提供了结构解释。

结论

本研究建立的集成平台结合了灵敏的荧光功能检测与AI驱动的结构分析，可实现胱氨酸尿症变异体的快速精准评估。该法无需放射性同位素，兼容常规微孔板读板设备，可嵌入现有变异解读流程，未来有潜力指导基因型个体化治疗，但尚需前瞻性临床验证。

Abstract

Objective

Cystine stones account for 1%‒2% of adult and up to 10% of pediatric kidney stones. They result from cystinuria

an autosomal recessive disorder caused by mutations in solute carrier family 3 member 1 (

SLC3A1

) and

SLC7A9

which encode the renal cystine transporter subunits. These mutations impair cystine reabsorption

raising urinary cystine levels and driving stone formation. Current diagnostic options remain limited in terms of detecting molecular dysfunctions. Thus

we aimed to develop a nonradioactive

cell-based method for the functional assessment of cystine tran

sporters and mutation-specific pathologies.

Methods

Using human embryonic kidney 293 (HEK293) cells transiently co-expressing wild-type or mutant

SLC3A1

and

SLC7A9

we developed an integrated approach that combined a selenocystine-based fluorescence uptake assay with AlphaFold3-based structural predictions to rapidly and accurately assess cystine transporter function and the molecular impact of genetic mutations.

Results

The affinity of the SLC3A1/SLC7A9 complex was comparably apparent for selenocystine (Michaelis constant

=(156.3±24.2) μmol/L) and cystine (literature

approximately 200 μmol/L). Using operational thresholds (mild

60%

moderate 20%‒60%

severe

20% residual activity)

the assay differentiated the functional impacts of eight clinically characterized variants

including

SLC7A9

A70V

A182T

G105R

R333W

V170M

A354T

and P482L

and

SLC3A1

M467T

with categorical assignments consistent with previously published radioisotope-based functional data. AlphaFold3 modeling

combined with molecular docking

provides mechanistic interpretations of the dysfunction observed in the P482L and A354T mutants.

Conclusions

The integrated approach employed in this work

which combines a sensitive selenocystine fluorescence assay with artificial intelligence (AI)-powered structural analysis

enables the rapid

precise diagnosis of cystinuria variants. This platform is compatible with standard microplate-reader infrastructure and offers potential utility in variant-interpretation pipelines and future genotype-guided therapeutic decision-making

pending prospective clinical validation.

关键词

Keywords

references

Abramson J , Adler J , Dunger J , et al. , 2024 . Accurate structure prediction of biomolecular interactions with AlphaFold 3 . Nature , 630 ( 8016 ): 493 - 500 . https://doi.org/10.1038/s41586-024-07487-w https://doi.org/10.1038/s41586-024-07487-w

Bartoccioni P , Rius M , Zorzano A , et al. , 2008 . Distinct classes of trafficking rBAT mutants cause the type I cystinuria phenotype . Hum Mol Genet , 17 ( 12 ): 1845 - 1854 . https://doi.org/10.1093/hmg/ddn080 https://doi.org/10.1093/hmg/ddn080

Bertran J , Werner A , Moore ML , et al. , 1992 . Expression cloning of a cDNA from rabbit kidney cortex that induces a single transport system for cystine and dibasic and neutral amino acids . Proc Natl Acad Sci USA , 89 ( 12 ): 5601 - 5605 . https://doi.org/10.1073/pnas.89.12.5601 https://doi.org/10.1073/pnas.89.12.5601

Chairoungdua A , Segawa H , Kim JY , et al. , 1999 . Identification of an amino acid transporter associated with the cystinuria-related type II membrane glycoprotein . J Biol Chem , 274 ( 41 ): 28845 - 28848 . https://doi.org/10.1074/jbc.274.41.28845 https://doi.org/10.1074/jbc.274.41.28845

Chillarón J , Font-Llitjós M , Fort J , et al. , 2010 . Pathophysiology and treatment of cystinuria . Nat Rev Nephrol , 6 ( 7 ): 424 - 434 . https://doi.org/10.1038/nrneph.2010.69 https://doi.org/10.1038/nrneph.2010.69

Copelovitch L , 2012 . Urolithiasis in children: medical approach . Pediatr Clin North Am , 59 ( 4 ): 881 - 896 . https://doi.org/10.1016/j.pcl.2012.05.009 https://doi.org/10.1016/j.pcl.2012.05.009

Feliubadaló L , Font M , Purroy J , et al. , 1999 . Non-type I cystinuria caused by mutations in SLC7 A9, encoding a subunit (b 0,+ AT) of rBAT . Nat Genet, 23 : 52 - 57 . https://doi.org/10.1038/12652 https://doi.org/10.1038/12652

Font M , Feliubadaló L , Estivill X , et al. , 2001 . Functional analysis of mutations in SLC7A9 , and genotype‒phenotype correlation in non-Type I cysti nuria . Hum Mol Genet , 10 ( 4 ): 305 - 316 . https://doi.org/10.1093/hmg/10.4.305 https://doi.org/10.1093/hmg/10.4.305

Jumper J , Evans R , Pritzel A , et al. , 2021 . Highly accurate protein structure prediction with AlphaFold . Nature , 596 ( 7873 ): 583 - 589 . https://doi.org/10.1038/s41586-021-03819-2 https://doi.org/10.1038/s41586-021-03819-2

Kalaiyarasan G , Hemlata C , Joseph J , 2019 . Fluorescence turn-on, specific detection of cystine in human blood plasma and urine samples by nitrogen-doped carbon quantum dots . ACS Omega , 4 ( 1 ): 1007 - 1014 . https://doi.org/10.1021/acsomega.8b03187 https://doi.org/10.1021/acsomega.8b03187

Lee Y , Wiriyasermkul P , Kongpracha P , et al. , 2022 . Ca 2+ -mediated higher-order assembly of heterodimers in amino acid transport system b 0,+ biogenesis and cystinuria . Nat Commun , 13 : 2708 . https://doi.org/10.1038/s41467-022-30293-9 https://doi.org/10.1038/s41467-022-30293-9

Mizoguchi KI , Cha SH , Chairoungdua A , et al. , 2001 . Human cystinuria-related transporter: localization and functional characterization . Kidney Int , 59 ( 5 ): 1821 - 1833 . https://doi.org/10.1046/j.1523-1755.2001.0590051821.x https://doi.org/10.1046/j.1523-1755.2001.0590051821.x

Nagamori S , Wiriyasermkul P , Guarch ME , et al. , 2016 . Novel cystine transporter in renal proximal tubule identified as a missing partner of cystinuria-related plasma membrane protein rBAT/SLC3A1 . Proc Natl Acad Sci USA , 113 ( 3 ): 775 - 780 . https://doi.org/10.1073/pnas.1519959113 https://doi.org/10.1073/pnas.1519959113

Olschok K , Vester U , Lahme S , et al. , 2018 . No evidence for point mutations in the novel renal cystine transporter AGT1/SLC7A13 contributing to the etiology of cystinuria . BMC Nephrol , 19 : 278 . https://doi.org/10.1186/s12882-018-1080-5 https://doi.org/10.1186/s12882-018-1080-5

Reig N , Chillarón J , Bartoccioni P , et al. , 2002 . The light subunit of system b 0,+ is fully functional in the absence of the heavy subunit . EMBO J , 21 ( 18 ): 4906 - 4914 . https://doi.org/10.1093/emboj/cdf500 https://doi.org/10.1093/emboj/cdf500

Servais A , Thomas K , Dello Strologo L , et al. , 2021 . Cystinuria: clinical practice recommendation . Kidney Int , 99 ( 1 ): 48 - 58 . https://doi.org/10.1016/j.kint.2020.06.035 https://doi.org/10.1016/j.kint.2020.06.035

Shigeta Y , Kanai Y , Chairoungdua A , et al. , 2006 . A novel missense mutation of SLC7A9 frequent in Japanese cystinuria cases affecting the C-terminus of the transporter . Kidney Int , 69 ( 7 ): 1198 - 1206 . https://doi.org/10.1038/sj.ki.5000241 https://doi.org/10.1038/sj.ki.5000241

Shimomura T , Hirakawa N , Ohuchi Y , et al. , 2021 . Simple fluorescence assay for cystine uptake via the xCT in cells using selenocystine and a fluorescent probe . ACS Sens , 6 ( 6 ): 2125 - 2128 . https://doi.org/10.1021/acssensors.1c00496 https://doi.org/10.1021/acssensors.1c00496

Stenson PD , Ball EV , Mort M , et al. , 2003 . Human Gene Mutation Database (HGMD ® ): 2003 update . Hum Mutat , 21 ( 6 ): 577 - 581 . https://doi.org/10.1002/humu.10212 https://doi.org/10.1002/humu.10212

Thomas K , Wong K , Withington J , et al. , 2014 . Cystinuria—a urologist’s perspective . Nat Rev Urol , 11 ( 5 ): 270 - 277 . https://doi.org/10.1038/nrurol.2014.51 https://doi.org/10.1038/nrurol.2014.51

Wells RG , Hediger MA , 1992 . Cloning of a rat kidney cDNA that stimulates dibasic and neutral amino acid transport and has sequence similarity to glucosidases . Proc Natl Acad Sci USA , 89 ( 12 ): 5596 - 5600 . https://doi.org/10.1073/pnas.89.12.5596 https://doi.org/10.1073/pnas.89.12.5596

Wu D , Grund TN , Welsch S , et al. , 2020 . Structural basis for amino acid exchange by a human heteromeric amino acid transporter . Proc Natl Acad Sci USA , 117 ( 35 ): 21281 - 21287 . https://doi.org/10.1073/pnas.2008111117 https://doi.org/10.1073/pnas.2008111117

Yan RH , Li YN , Shi Y , et al. , 2020 . Cryo-EM structure of the human heteromeric amino acid transporter b 0,+ AT-rBAT . Sci Adv , 6 ( 16 ): eaay6379 . https://doi.org/10.1126/sciadv.aay6379 https://doi.org/10.1126/sciadv.aay6379

Zhang H , Lan JJ , Wang HJ , et al. , 2024 . AlphaFold2 in biomedical research: facilitating the development of diagnostic strategies for disease . Front Mol Biosci , 11 : 1414916 . https://doi.org/10.3389/fmolb.2024.1414916 https://doi.org/10.3389/fmolb.2024.1414916

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