Jin Cheng Long, Oh Jang-Hee, Han Mira, Shin Min Kyeong, Yao Cheng, Park Chi-Hyun, Jin Zhe Hu, Chung Jin Ho
Department of Dermatology, Seoul National University College of Medicine, Seoul, Republic of Korea; Department of Dermatology, Yanbian University Hospital, Yanji, Jilin, China; Laboratory of Cutaneous Aging Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea; Institute of Human-Environment Interface Biology, Medical Research Center, Seoul National University, Seoul, Republic of Korea.
Department of Dermatology, Seoul National University College of Medicine, Seoul, Republic of Korea; Laboratory of Cutaneous Aging Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea; Institute of Human-Environment Interface Biology, Medical Research Center, Seoul National University, Seoul, Republic of Korea.
J Dermatol Sci. 2015 Jul;79(1):20-9. doi: 10.1016/j.jdermsci.2015.03.018. Epub 2015 Apr 10.
Biglycan (BGN) is a proteoglycan composed of a 42-kDa core protein and two glycosaminoglycan (GAG) chains, and known to be involved in structural, space-filling functions and many physiological regulations in the skin.
To investigate ultraviolet (UV) irradiation-induced changes of BGN protein and its GAG chain synthesis in cultured human dermal fibroblasts.
UV irradiation-induced or xylosyltransferase (XYLT) 1 siRNA-mediated smaller-sized protein bands detected by Western blot using BGN antibodies were identified as monoglycosylated forms of BGN, using BGN siRNA-mediated knockdown and chondroitinase ABC (ChABC). Differential activity of XYLT1 and 2 on BGN core protein was investigated by size shift of S42A- and S47A-BGN mutants to core protein size caused by XYLT1 siRNA transfection or UV irradiation.
After UV irradiation, intact form of BGN protein (I-BGN) and core protein form were reduced in cultured fibroblasts, but other smaller-sized bands were observed to be increased. These smaller-sized ones were reduced by transfection of BGN siRNA, and shifted to the core protein size by treatment with ChABC, suggesting that they are defectively-glycosylated forms of BGN (D-BGN) protein. UV irradiation also decreased mRNA expression levels of XYLT1 and 2, which are responsible for initiation of GAG chain synthesis. UV-mediated reduction of XYLT1 expression was much stronger than that of XYLT2. Furthermore, siRNA-mediated down-regulation of XYLT1 resulted in the increase of D-BGN and the decrease of I-BGN, while down-regulation of XYLT2 resulted in no change of D-BGN and I-BGN, suggesting that the XYLT1 may react with both GAG-attaching serine sites of BGN; however, XYLT2 may prefer to react one of them. Another dermatan sulfate (DS) proteoglycan, decorin, showed no or a little change of its molecular weight by UV irradiation or XYLT1 siRNA transfection, suggesting that DS synthesis may not be a critical factor in formation of D-BGN. Co-transfection with XYLT1, 2 siRNAs and wild-type or mutant forms of BGN overexpression vectors revealed that S42A-BGN showed size reduction to core protein size by XYLT1 downregulation, but S47A-BGN did not, suggesting that XYLT2 can react only with S42 on BGN core protein. With UV irradiation, both S42A-BGN and S47A-BGN showed size reduction, which is probably because UV-caused downregulation of both XYLTs and overexpression condition resulted in incomplete glycosylation and secretion.
UV irradiation-induced increase of BGN monoglycosylated forms in cultured human dermal fibroblasts is resulted from dominance of XYLT2 activity, which acts only at S42 on BGN core protein, caused by UV-mediated stronger reduction of XYLT1.
双糖链蛋白聚糖（BGN）是一种蛋白聚糖，由一个42 kDa的核心蛋白和两条糖胺聚糖（GAG）链组成，已知其参与皮肤的结构、填充功能以及许多生理调节过程。
研究紫外线（UV）照射诱导培养的人真皮成纤维细胞中BGN蛋白及其GAG链合成的变化。
使用BGN抗体通过蛋白质印迹法检测到的UV照射诱导或木糖基转移酶（XYLT）1小干扰RNA（siRNA）介导的较小尺寸蛋白条带，利用BGN siRNA介导的敲低和软骨素酶ABC（ChABC）鉴定为BGN的单糖基化形式。通过XYLT1 siRNA转染或UV照射导致的S42A-和S47A-BGN突变体向核心蛋白大小的大小偏移，研究XYLT1和2对BGN核心蛋白的差异活性。
UV照射后，培养的成纤维细胞中BGN蛋白的完整形式（I-BGN）和核心蛋白形式减少，但观察到其他较小尺寸的条带增加。这些较小尺寸的条带通过BGN siRNA转染而减少，并通过ChABC处理转移到核心蛋白大小，表明它们是BGN（D-BGN）蛋白的糖基化缺陷形式。UV照射还降低了负责GAG链合成起始的XYLT1和2的mRNA表达水平。UV介导的XYLT1表达降低比XYLT2更强。此外，siRNA介导的XYLT1下调导致D-BGN增加和I-BGN减少，而XYLT2下调导致D-BGN和I-BGN无变化，表明XYLT1可能与BGN的两个GAG连接丝氨酸位点都发生反应；然而，XYLT2可能更倾向于与其中一个发生反应。另一种硫酸皮肤素（DS）蛋白聚糖，核心蛋白聚糖，在UV照射或XYLT1 siRNA转染后其分子量没有变化或仅有轻微变化，表明DS合成可能不是D-BGN形成的关键因素。XYLT1、2 siRNAs与野生型或突变型BGN过表达载体共转染显示，XYLT1下调导致S42A-BGN大小减小至核心蛋白大小，但S47A-BGN没有，表明XYLT2只能与BGN核心蛋白上的S42发生反应。UV照射后，S42A-BGN和S47A-BGN均显示大小减小，这可能是因为UV导致的两种XYLTs下调和过表达条件导致糖基化和分泌不完全。
培养的人真皮成纤维细胞中UV照射诱导BGN单糖基化形式增加是由于UV介导的XYLT1更强降低导致XYLT2活性占主导，而XYLT2仅作用于BGN核心蛋白上的S42。
